EP2267023A2 - KDR peptides and vaccines comprising the same - Google Patents
KDR peptides and vaccines comprising the same Download PDFInfo
- Publication number
- EP2267023A2 EP2267023A2 EP10010175A EP10010175A EP2267023A2 EP 2267023 A2 EP2267023 A2 EP 2267023A2 EP 10010175 A EP10010175 A EP 10010175A EP 10010175 A EP10010175 A EP 10010175A EP 2267023 A2 EP2267023 A2 EP 2267023A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptides
- cells
- hla
- seq
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 16
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 title description 111
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 271
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 170
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 claims abstract description 128
- 150000001413 amino acids Chemical class 0.000 claims abstract description 74
- 229940024606 amino acid Drugs 0.000 claims description 49
- 108091007433 antigens Proteins 0.000 claims description 41
- 239000000427 antigen Substances 0.000 claims description 40
- 102000036639 antigens Human genes 0.000 claims description 40
- 108010013476 HLA-A24 Antigen Proteins 0.000 claims description 31
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 15
- 230000033115 angiogenesis Effects 0.000 claims description 15
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 14
- 210000001808 exosome Anatomy 0.000 claims description 12
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 11
- 230000002401 inhibitory effect Effects 0.000 claims description 11
- 229930182817 methionine Natural products 0.000 claims description 11
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 8
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 8
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 8
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 8
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- 229960000310 isoleucine Drugs 0.000 claims description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 49
- 239000003814 drug Substances 0.000 abstract description 20
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 3
- 210000004027 cell Anatomy 0.000 description 166
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 106
- 230000001472 cytotoxic effect Effects 0.000 description 63
- 231100000433 cytotoxic Toxicity 0.000 description 54
- 235000001014 amino acid Nutrition 0.000 description 47
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 42
- 230000000694 effects Effects 0.000 description 31
- 241000699670 Mus sp. Species 0.000 description 29
- 230000000977 initiatory effect Effects 0.000 description 29
- 210000004443 dendritic cell Anatomy 0.000 description 28
- 210000000612 antigen-presenting cell Anatomy 0.000 description 25
- 238000000034 method Methods 0.000 description 25
- 238000003556 assay Methods 0.000 description 23
- 230000003013 cytotoxicity Effects 0.000 description 16
- 231100000135 cytotoxicity Toxicity 0.000 description 16
- 210000002889 endothelial cell Anatomy 0.000 description 15
- 210000004204 blood vessel Anatomy 0.000 description 14
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 13
- 241000699660 Mus musculus Species 0.000 description 13
- 239000011651 chromium Substances 0.000 description 13
- 229910052804 chromium Inorganic materials 0.000 description 13
- 238000011830 transgenic mouse model Methods 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 12
- 230000000638 stimulation Effects 0.000 description 11
- 238000009566 cancer vaccine Methods 0.000 description 10
- 229940022399 cancer vaccine Drugs 0.000 description 10
- 210000001072 colon Anatomy 0.000 description 10
- 230000001939 inductive effect Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000002560 therapeutic procedure Methods 0.000 description 10
- 206010009944 Colon cancer Diseases 0.000 description 9
- 102100037850 Interferon gamma Human genes 0.000 description 9
- 108010074328 Interferon-gamma Proteins 0.000 description 9
- 208000029742 colonic neoplasm Diseases 0.000 description 9
- 230000002062 proliferating effect Effects 0.000 description 9
- 241000701161 unidentified adenovirus Species 0.000 description 9
- 238000002255 vaccination Methods 0.000 description 9
- 239000012981 Hank's balanced salt solution Substances 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 230000001173 tumoral effect Effects 0.000 description 8
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 7
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 7
- 108010075704 HLA-A Antigens Proteins 0.000 description 6
- 102000011786 HLA-A Antigens Human genes 0.000 description 6
- 108091008605 VEGF receptors Proteins 0.000 description 6
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 231100000065 noncytotoxic Toxicity 0.000 description 6
- 210000005259 peripheral blood Anatomy 0.000 description 6
- 239000011886 peripheral blood Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 238000011725 BALB/c mouse Methods 0.000 description 5
- 102000000588 Interleukin-2 Human genes 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000001185 bone marrow Anatomy 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 5
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000036961 partial effect Effects 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- 108010004729 Phycoerythrin Proteins 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 210000001015 abdomen Anatomy 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 230000006427 angiogenic response Effects 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000005917 in vivo anti-tumor Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 238000010647 peptide synthesis reaction Methods 0.000 description 4
- 238000011321 prophylaxis Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 201000001320 Atherosclerosis Diseases 0.000 description 3
- 206010012689 Diabetic retinopathy Diseases 0.000 description 3
- 208000001382 Experimental Melanoma Diseases 0.000 description 3
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 201000004681 Psoriasis Diseases 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 108010081641 arginyl-phenylalanyl-valyl-prolyl-aspartyl-glycyl-asparagyl-arginyl-isoleucine Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000010432 diamond Substances 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 108091005601 modified peptides Proteins 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- BJHCYTJNPVGSBZ-YXSASFKJSA-N 1-[4-[6-amino-5-[(Z)-methoxyiminomethyl]pyrimidin-4-yl]oxy-2-chlorophenyl]-3-ethylurea Chemical compound CCNC(=O)Nc1ccc(Oc2ncnc(N)c2\C=N/OC)cc1Cl BJHCYTJNPVGSBZ-YXSASFKJSA-N 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 101150013553 CD40 gene Proteins 0.000 description 2
- 241000725101 Clea Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 102100022297 Integrin alpha-X Human genes 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 2
- 230000016571 aggressive behavior Effects 0.000 description 2
- 210000004712 air sac Anatomy 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000006287 biotinylation Effects 0.000 description 2
- 238000007413 biotinylation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229910003460 diamond Inorganic materials 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000002433 mononuclear leukocyte Anatomy 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000005747 tumor angiogenesis Effects 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102100035793 CD83 antigen Human genes 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 206010061825 Duodenal neoplasm Diseases 0.000 description 1
- 238000011510 Elispot assay Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 1
- 108010086377 HLA-A3 Antigen Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101100395310 Homo sapiens HLA-A gene Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- 101001043807 Homo sapiens Interleukin-7 Proteins 0.000 description 1
- 101800000324 Immunoglobulin A1 protease translocator Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101000746372 Mus musculus Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001002703 Mus musculus Interleukin-4 Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010054184 Small intestine carcinoma Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 1
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 201000000312 duodenum cancer Diseases 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000046157 human CSF2 Human genes 0.000 description 1
- 102000055229 human IL4 Human genes 0.000 description 1
- 102000052622 human IL7 Human genes 0.000 description 1
- 102000055590 human KDR Human genes 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464403—Receptors for growth factors
- A61K39/464409—Vascular endothelial growth factor receptors [VEGFR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/50—Colon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/57—Skin; melanoma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to novel peptides that are extremely useful as cancer vaccines, and pharmaceuticals that comprise these peptides, for treating and preventing tumors.
- Tumor-rejection antigen genes have mainly been identified for malignant melanomas, and accordingly, cancer immunotherapies utilizing these genes are developed.
- cancer vaccine therapy that induces tumor-specific CD8-positive T cells in vivo has received attention, and is being applied to various clinical applications.
- the mechanism by which peptides consisting of approximately ten amino acid residues activate T cells via the Class I pathway, by,the assistance of various costimulatory molecules, to induce tumor-specific cytotoxic T cells (CTLs) was also elucidated.
- CTLs cytotoxic T cells
- VEGFR immunotolerance to VEGFR
- Plate, Millauer, and Risau et al confirmed that VEGFRs were strongly expressed in the endothelial cells of tumoral tissues.
- the immune response to autoantigens such as CEA and HER/neu which are also expressed in normal cells, is not necessarily one of immunotolerance.
- the present inventors reasoned that VEGFRs may be used as targets for cancer vaccine therapy.
- the present inventors focused on possible cancer vaccine therapies that target VEGFR2 (KDR/flk-1; referred to below as KDR), where VEGFR2 is strongly expressed in tumoral tissue endothelial cells, and is thought be involved in the proliferation of endothelial cells on the VEGF signal. Furthermore, the present inventors screened for peptides that can be effectively used as vaccines, examining their specificity, and completing this invention.
- the present invention provides [1] to [22] as follows.
- the present inventors first considered that various proteins are presented in vivo on antigen presenting cells after being degraded to 9-mer peptides (nonapeptides), and examined the binding affinity of 9-mer or 10-mer partial peptides of KDR proteins to HLA antigens, which are human major histocompatibiliy antigens (MHC antigens).
- HLA antigens which are human major histocompatibiliy antigens (MHC antigens).
- MHC antigens human major histocompatibiliy antigens
- the amino acid sequence of human KDR protein is well known, disclosed by US Patent No. 5,861,301 for example, and can be easily obtained by one skilled in the art.
- the 9-mer and 10-mer peptides can be obtained by synthesizing peptides initiated from any position, based on the full-length amino acid sequence of the obtained KDR protein.
- Peptides can be synthesized according to conventional methods used in peptide chemistry. Commonly used synthesis methods are described, for example, in literatures such as Peptide Synthesis, Interscience, New York, 1966 ; The Proteins, vol.
- Binding with HLA antigens can be measured by isolating cells that comprise HLA antigens on their cell surface, such as dendritic cells, and using conventional methods to measure the binding of peptides to cells.
- binding affinity with HLA antigens can be measured as described, for example, in Parker, K. C., J. Immunol., 152, 1994 ; and Nukaya, I., Int. J. Cancer, 80, 1999 .
- A-24 type and A-02 type antigens are preferably used as the HLA antigens. More preferably, subtypes such as A-2402 and A-0201 are used.
- a peptide can be appropriately selected to have a high level of binding affinity to that antigen, or level of cytotoxic T cell (CTL) inducibility upon antigen presentation.
- CTL cytotoxic T cell
- substitution or addition of one, two, or several amino acids may be performed based on the amino acid sequence of the naturally occurring KDR partial peptide.
- the term "several” means five or less, or preferably three or less.
- sequence regularity of the peptides displayed by binding to HLA antigens is already known ( J. Immunol., 152, 3913, 1994 ; Immunogenetics. 41:178, 1995 ; J. Immunol. 155:4307, 1994 ). Thus, modifications based on this regularity can also be performed on the obtained peptides.
- peptides in which the second amino acid from the N terminus is substituted with phenylalanine, tyrosine, methionine, or tryptophan, and in which the C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine may also be preferably used as peptides with high HLA-24 binding affinity.
- peptides in which the second amino acid from the N terminus is substituted with leucine or methionine, and in which C-terminal amino acid is substituted with valine or leucine may be preferably used as peptides with high HLA-0201 binding affinity.
- one to two amino acids may also be added to the N and/or C terminus of the peptides.
- a peptide sequence is identical to a portion of an amino acid sequence of an endogenous or exogenous protein with a different function, it may cause side effects such as autoimmune diseases or allergic symptoms against specific substances. Therefore, it is preferable to use available databases to carry out homology searches, to avoid situations in which a sequence matches the amino acid sequence of another protein. Furthermore, if homology searches reveal that no peptides comprising even a one- or two- amino acid difference exist, there is no risk of such problems caused by modifications of the above-mentioned amino acid sequences for increasing the binding affinity with HLA antigens and/or the CTL inducibility.
- peptides with high binding affinity to the HLA antigens are expected to be highly effective as cancer vaccines.
- Confirmation of CTL inducibility is carried out by inducing antigen-presenting cells comprising human MHC antigens (such as B-lymphocytes, macrophages, and dendritic cells), more specifically dendritic cells derived from human peripheral blood mononuclear leukocytes; stimulating the cells with the peptides; mixing the cells with CD8-positive cells; and then measuring cytotoxicity against the target cells.
- human MHC antigens such as B-lymphocytes, macrophages, and dendritic cells
- transgenic animals generated to express a human HLA antigen may be used (for example, those described in Hum. Immunol. 2000 Aug.; 61(8):764-79 Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response., BenMohamed L., Krishnan R., Longmate J., Auge C., Low L., Primus J., and Diamond DJ.).
- cytotoxicity can be calculated from the radioactivity released from the target cells.
- the activity can be examined by measuring the IFN- ⁇ produced and released by CTL in the presence of antigen-presenting cells that have immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN- ⁇ monoclonal antibodies.
- the present invention further provides peptides with cytotoxic T cell inducibility, wherein one, two, or several amino acids are substituted with or added to the amino acid sequence of SEQ ID NO: 2, 3, 6, 8, 11, or 12.
- Amino acid sequences consisting of nine amino acids of SEQ ID NOs: 2, 3, 5, 8, 11, and 12, in which one, two, or several amino acids are substituted or added, may have CTL inducibility, as long as they do not match the amino acid sequence of another protein.
- the second amino acid from the N terminus is preferably substituted to phenylalanine, tyrosine, methionine, or tryptophan
- the C-terminal amino acid is preferably substituted to phenylalanine, leucine, isoleucine, tryptophan, or methionine; or one or two amino acids are added to the N terminus and/or C terminus.
- the present invention also provides peptides with cytotoxic T cell inducibility, wherein one, two, or several amino acids are substituted or added to the amino acid sequence of SEQ ID NOs: 29, 30, 33, 34, 40, or 46.
- Amino acid sequences consisting of nine or ten amino acids of SEQ ID NOs: 29, 30, 33, 34, 40, and 46, in which one, two, or several amino acids are substituted or added, may have CTL inducibility as long as they do not match the amino acid sequence of another protein.
- the second amino acid from the N terminus is preferably substituted to leucine or methionine, or the C-terminal amino acid is preferably substituted to valine or leucine; or one or two amino acids are added to the N terminus and/or C terminus.
- An example of such a modified peptide is the peptide of SEQ ID NO: 30, in which the second amino acid from the N terminus is substituted to leucine (SEQ ID NO: 54), but this example is not limiting. CTL clones obtained on stimulation with these modified peptides can recognize the original peptides, and cause damage.
- the peptides of this invention can be used alone or in combinations of two or more, as cancer vaccines capable of inducing CTL in vivo.
- the peptides are presented at a high density on the HLA antigens of antigen-presenting cells.
- CTLs that specifically react with the complexes formed between the displayed peptides and the HLA antigens are induced, increasing aggression against vascular endothelial cells in the tumor cells to be targeted.
- antigen-presenting cells on whose cell surface the peptides of this invention are immobilized can be obtained by deriving dendritic cells from a subject, and stimulating them with the peptides of this invention.
- the obtained antigen-presenting cells are re-administered to the subjects to induce CTL in the subjects.
- aggression towards the target cells can be increased.
- the present invention provides pharmaceuticals for treating tumors or preventing proliferation, metastasis, and such of tumors, where the pharmaceuticals comprise one or more peptides of this invention.
- Angiogenesis at pathologic sites is closely associated with not only tumors, but also with diseases such as diabetic retinopathy, chronic rheumatoid arthritis, psoriasis, and atherosclerosis, and the metastasis of solid tumors ( Folkman, J., Nature Med. 1:27-31 (1995 ); Bicknell, R., Harris, A.L., Curr. Opin. Oncol. 8:60-65 (1996 )). Therefore, the peptides of this invention can be used to treat tumors, diseases such as diabetic retinopathy, chronic rheumatoid arthritis, psoriasis, atherosclerosis, and metastasis of solid tumors.
- the peptides of this invention were confirmed to inhibit the formation of tortuous blood vessels, which are morphologically different from normal blood vessels, and which are formed in malignant tumoral tissues.
- the results of analyzing wound healing and fertility in vaccinated mice also confirmed that the peptides do not have an adverse effect on normal physiological angiogenesis.
- CTL clones that recognize the peptides of this invention cytotoxicity against non-proliferative or proliferative endothelial cells was tested in vitro. These clones showed stronger activity towards proliferative endothelial cells than non-proliferative endothelial cells. More specifically, they can function very specifically for disorders involving proliferative endothelial cells, and particularly cancer.
- the peptides of this invention In vivo and in vitro stimulation of dendritic cells by the peptides of this invention can be easily performed by exposing the cells to a high concentration of the peptides, which causes these peptides to replace the peptides originally immobilized on the cells. Therefore, the peptides used in this invention must have at least a certain level of binding affinity to HLA antigens.
- the pharmaceuticals of this invention may be directly administered as the peptides of this invention themselves, or may be administered as pharmaceutical compositions that have been formulated by conventional formulation methods.
- the pharmaceuticals of this invention can appropriately include, in addition to the peptides of this invention, carriers, excipients, and such that are ordinarily used for pharmaceuticals, without particular limitations.
- the pharmaceuticals of this invention can be used for treatment and prevention of various tumors, such as gastric cancer, duodenal cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and brain tumors.
- the peptides of this invention do not target the tumor cells themselves, but target the endothelial cells of blood vessels that are newly formed in tumoral tissues. Therefore, a wide variety of tumors can be treatment targets, and the pharmaceuticals of this invention are not particularly limited in their use.
- compositions for treating and/or preventing tumors which comprise a peptide of this invention as an active ingredient, can be administered with adjuvants so that cellular immunity is induced effectively; can be administered with other active ingredients such as antitumor agents; and can be administered in granular forms.
- adjuvants described in the literature ( Clin. Microbiol. Rev., 7:277-289, 1994 ) or such are applicable.
- the pharmaceuticals of this invention can be administered as liposome formulations, as granular formulations bound to beads of a few micrometers in diameter, and as formulations to which lipids are bound. Administration methods may be carried out, for example, orally, intradermally, or subcutaneously, or through intravenous injection, or such.
- Systemic administration or local administration to the vicinity of the target tumor may be applicable.
- Doses of the peptides of this invention can be adjusted appropriately, depending on the disease to be treated, age and weight of the patients, administration methods, and such. Ordinarily, 0.001 mg to 1,000 mg, preferably 0.001 mg to 1,000 mg, more preferably 0.1 mg to 10 mg, of the peptides of this invention are preferably administered once in a few days to a few months. One skilled in the art can appropriately select suitable doses.
- the present invention provides intracellular vesicles which present complexes formed between the peptides of this invention and HLA antigens on their surface. These intracellular vesicles are called exosomes.
- Exosomes can be prepared, for example, according to the methods specifically described in Published Japanese Translation of International Publication Nos. Hei 11-510507 and 2000-512161 . Exosomes can preferably be prepared using antigen-presenting cells obtained from subjects who are to be the target of therapy or prophylaxis.
- the exosomes of this invention can be inoculated as cancer vaccines, as for the peptides of this invention.
- HLA antigens to be used must match that of the subject in need of therapy and/or prophylaxis.
- HLA-A24 or HLA-A02 particularly HLA-A2402 or HLA-0201, is often appropriate.
- the present invention also provides methods for inducing antigen-presenting cells using the peptides of this invention.
- the antigen-presenting cells can be induced by inducing dendritic cells from peripheral blood monocytes; and then contacting (stimulating) them with the peptides of this invention, in vitro or in vivo.
- Administering the peptides of this invention to subjects induces in the body of the subject antigen-presenting cells to which the peptides of this invention are immobilized.
- the peptides of this invention can be immobilized to the antigen-presenting cells to be administered to the subject as a vaccine.
- the present invention also provides methods for inducing antigen-presenting cells with a high level of cytotoxic T cell inducibility, wherein said methods comprise the in vitro introduction to an antigen-presenting cell of a gene that comprises a polynucleotide encoding a peptide of this invention.
- the genes to be introduced may be in the form of DNA or RNA.
- the introduction methods may be various methods commonly used in the art, such as lipofection, electroporation, and calcium phosphate methods, without particular limitations. More specifically, the methods may be performed as described in, for example. Cancer Res., 56:5672, 1996 ; J. Immunol., 161:5607, 1998 ; J. Exp.
- the present invention provides methods for inducing CTLs using the peptides of this invention.
- CTLs are induced in the body of the subject, enhancing immunity against the angiogenic endothelial cells in the tumoral tissues.
- the methods may be used for ex vivo therapeutic methods, in which subject-derived antigen-presenting cells, CD8-positive cells, or peripheral blood mononuclear leukocytes are contacted (stimulated) in vitro with the peptides of this invention to induce CTLs, and then the cells are returned to the subject.
- the present invention provides cytotoxic T cells, that are induced using the peptides of this invention and then isolated.
- the cytotoxic T cells which have been induced by stimulation with antigen-presenting cells that present the peptides of this invention, are preferably derived from subjects to be the target of therapy and/or prophylaxis.
- the cytotoxic T cells can be administered alone or, for the purpose of antitumor effect, in combination with other drugs, including the peptides, exosomes and so on of this invention.
- the obtained cytotoxic T cells act specifically against target cells presenting the peptides of this invention, or preferably, against target cells presenting the same peptides used for induction.
- the target cells may be cells that endogenously express KDR, or cells forced to express KDR.
- cells that present the peptides of this invention on their cell surface due to stimulation by these peptides can also be targeted.
- the present invention also provides antigen-presenting cells that present complexes formed between HLA antigens and the peptides of this invention.
- the antigen-presenting cells that are obtained by contact with the peptides of this invention, or with nucleotides encoding the peptides of this invention, are preferably derived from subjects to be targeted for therapy and/or prophylaxis.
- the antigen-presenting cells can be administered as vaccines alone, or in combination with other drugs such as the peptides of this invention, exosomes, and cytotoxic T cells.
- CE652 (SEQ ID NO: 25) refers to one of the epitopes of tumor antigen CEA (carcinoembryonic antigen) that has been reported by Nukaya, I. (Int. J. Cancer 80, 1999). HIV peptides (ILKEPVHGV (SEQ ID NO: 55) and RYLRDQQLL (SEQ ID NO: 56)) were used as the negative control peptides.
- the peptides were synthesized according to standard solid-phase synthesis methods using Cleaved PepSet (Mimotope, San Diego, CA), and purified by reverse phase HPLC. The purity (>95%) and the type of peptide were individually determined using HPLC and mass spectrometry.
- Epstein-Barr virus (EBV)-immortalized B cell lines TISI (HLA-A24/24) and EHM (HLA-A3/3), were provided by Takara Shuzo Biotechnology Research Laboratory.
- PBMCs peripheral blood mononuclear lymphocytes
- the obtained PBMCs were left to stand for ten hours in a culture flask (Corning, 43072). After removing the suspended cells, the cells that adhered to the flask were cultured in AIM-V medium (Invitrogen) supplemented with 2% autoserum by adding 1,000 U/mL of human GM-CSF (provided by Kirin Brewery), and 1,000 U/mL of human IL-4 (Genzyme). Five days later, the cells were cultured for another 48 hours in 10 ⁇ g/mL of OK-432 (provided by Chugai Pharmaceutical Co., Ltd.), and were used as antigen-presenting cells for CTL induction.
- AIM-V medium Invitrogen
- human GM-CSF provided by Kirin Brewery
- human IL-4 Human IL-4
- DCs Dendritic cells
- FITC-labeled anti-ClassII, CD80, and CD86 antibodies were confirmed to be DCs by reaction with FITC-labeled anti-ClassII, CD80, and CD86 antibodies, and with PE-labeled anti-ClassI, CD11c, CD40 (all from Beckton-Dickinson), and CD83 (Immunotech) antibodies; followed by analysis of surface antigens by FACS-Calibur (Beckton-Dickinson) using Cell Quest software.
- Pre-induced DCs were pulsed for four hours at 20°C with twelve kinds of 9-mer peptides (SEQ ID NOs: 1 to 12) (20 ⁇ g/mL), that were predicted in Example 1 to have high binding affinity, in the presence of 3 ⁇ g/mL of ⁇ 2-microglobulin.
- the resulting DCs were mixed with CD8-positive cells selected by magnetic beads (Dynabeads M-450 and Detachabeads) from PBMC, at a ratio of 1:20 or 1:2, and then cultured in a 48-well plate (Corning), in the presence of 10 ng/mL of human IL-7 (Genzyme).
- Cytotoxic activity was evaluated by a four-hour 51 Cr release assay. A concentration of 20 ⁇ g/mL of the target cells were pulsed with peptides overnight. The target cells were labeled with 100 ⁇ Ci of Na 2 51 CrO 4 at 37°C for one hour, and then washed three times with RPMI1640. The target cells (1x10 4 /100 ⁇ L) and 100 ⁇ L of effector cells at various concentrations with a total volume of 200 ⁇ L were placed into a U-shaped 96-well microtiter plate (Corning), and cultured at 37°C in a CO 2 incubator for four hours. After culturing. 100 ⁇ L of supernatant was collected from each well, and measured using a ⁇ counter.
- CTL lines established in Example 2 were diluted to 0.3, 1, or 3 cells/well in a U-shaped 96-well plate. 7x 10 4 cells/well of allo-PBMC, 1x 10 4 cells/well of EHM, 30 ng/mL of anti-CD3 antibody, and 125 U/mL of IL-2 were added to each well, and AIM-V supplemented with 5% autoserum was added thereto, so that total concentration was 150 ⁇ L/well. Ten days later, 50 ⁇ L/well of IL-2-supplemented medium adjusted was added to a final IL-2 concentration of 125 U/mL. CTLs were functionally analyzed on the 14th day, and CTLs with activity were cultured on a large scale.
- Example 3 The degrees of cytotoxicity of the ten types of CTL clones established in Example 3 were examined by varying their ratio with the target cells (E:T ratio).
- C53-169 derived from the nonapeptide of SEQ ID NO: 8 (amino acid initiation position KDR169) showed the strongest cytotoxicity.
- HLA-tetramer was synthesized from HLA-A*2402 and the peptide of SEQ ID NO: 8 (amino acid initiation position KDR169). More specifically, plasmid vectors expressing the H chain (approximately 35 kDa) and L chain (approximately 11 kDa) were produced individually, and their recombinant protein was expressed using E. coli.
- the H-chain plasmid vector consists only of the extracellular domain, in which the carboxyl terminus was substituted with a biotinylation enzyme recognition sequence. The H chain and L chain form an HLA peptide complex by refolding with an antigenic peptide.
- HLA peptide complexes were biotinylated by a biotinylation enzyme, excess biotin was removed by gel filtration chromatography and ion exchange chromatography, and the biotinylated HLA peptide complexes were isolated and purified.
- HLA-tetramer was synthesized by reacting the obtained biotinylated HLA peptide complex with streptavidin (SA) at a mole ratio of 4:1 to form a tetramer.
- SA streptavidin
- SA-PE phycoerythrin
- CD8 molecule is known to bind to the 245th amino acid in the ⁇ 3 domain, which is alanine, with the exception of portions of HLA-A, B, and C. Therefore, the present inventors mutagenized the alanine in the HLA-tetramers to valine, to produce a mutant HLA-tetramer capable of selectively detecting CTLs with high avidity towards HLA peptide complexes, independent of the binding between the CD8 molecule and the ⁇ 3 domain.
- Established CTL clones were detected by the synthesized HLA-A24 KDR169-tetramer mutant. As a result, as shown in Fig. 2 , the established CTL clones were found to be strongly stained by the tetramer-positive and CD8-positive fractions, and to specifically recognize complexes formed between the peptides of this invention and HLA antigens. The signals in the tetramer-negative and CD8-negative fractions are due to feeder cells.
- CEA588 (SEQ ID NO: 53) refers to one of the epitopes of tumor antigen CEA (carcinoembryonic antigen) reported by Tanaka, H. et al. (poster presentation, AACR #3669, vol. 42, p681-682, March 2001 ).
- CTLs were induced by a vaccination method, using the 16 types of 9-mer and 10-mer peptides (SEQ ID NO: 26 to 41) predicted in Example 6 on transgenic mice that express human HLA (A0201) ( Hum. Immunol. 2000 Aug.;61(8):764-79 Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLAA*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response., BenMohamed L., Krishnan R., Longmate J., Auge C., Low L., Primus J., and Diamond DJ.).
- mice Six- to eight-week old BALB/C(H-2 d ) mice were obtained from CLEA Japan.
- A2/Kb transgenic mice (TGM) were provided by F. Jim Primus, Ph. D. at Vanderbilt-Ingram Cancer Center.
- T2 cells (TAP-deficient and HLA-A*0201-positive cells) were provided by Prof. H. Shiku at Mie University's Third Department of Internal Medicine.
- Bone marrow was collected from mouse femur and tibia, and lymphocytes and granulocytes were removed using anti-CD4, CD8, Gr-1 antibodies (Beckton-Dickinson), anti-B220 antibody (Bioscience) and rabbit complement (PeL-Freez). The obtained cells were cultured in a 6-well plate. Suspended cells were collected the following day to culture in RPMI1640 (Invitrogen) medium supplemented with 10% FBS in another 6-well plate, to which 1,000 U/mL of mouse GM-CSF (provided by Kirin Brewery) and 1,000 U/mL of mouse IL-4 (PEPRO TECH) were added.
- RPMI1640 Invitrogen
- mice 100 ⁇ g of each peptide, 140 ⁇ g of HbcAg120-140 helper peptide, and 100 ⁇ L of IFA were mixed (200 ⁇ L in total). The mixture was administered subcutaneously to the right abdomen on day 0, and to the left abdomen on day 11.
- the spleens of the vaccinated mice were collected on day 21, erythrocytes were hemolytically removed using red lysis buffer (Sigma), and a portion of those cells were used as responder cells for IFN- ⁇ ELISPOT assay.
- the remaining cells were placed into a 24-well plate (Corning) at 6x 10 6 cells/well with feeder cells at a ratio of 3:1, and stimulated again. Their cytotoxicity was measured five days later.
- Erythrocyte-free splenocytes which were derived from syngeneic mice, were cultured for three days in the presence of 25 ⁇ g/mL of lipopolysaccharide (LPS) to use as feeder cells.
- LPS lipo
- the IFN- ⁇ ELISPOT method was used to evaluate CTL inducibility according to SPOTs produced by IFN- ⁇ producing cells.
- Flat-bottomed 96-well multiscreen plate MAHA S45 (Millipore) was treated at 4°C overnight with anti-mouse IFN- ⁇ monoclonal antibody (Pharmingen).
- the plate washed with PBS containing 0.05% Tween 20, and then reacted with a blocking buffer at room temperature for two hours. Thereafter, peptide-pulsed T2 cells and unpulsed T2 cells were added to each well at 10 5 cells/100 ⁇ L.
- mice The splenocytes of vaccinated mice were further added to each well at a maximum of 4x 10 6 cells/100 ⁇ L (200 ⁇ L in total), and cultured overnight at 37°C. The next day, each well was washed, and reacted for two hours with biotinylated rat anti-mouse IFN- ⁇ antibodies (Pharmingen). After washing the plate thoroughly, Extravidin was added to each well and reacted at room temperature for two hours. After washing, alkaline phosphatase conjugate substrate (BIO-RAD) was added to the wells, which were left to stand at room temperature for five minutes. The blue spots resulting from IFN- ⁇ production were measured using KS ELISPOT compact release (Carl Zeiss).
- Bone marrow was collected from six- to eight-week old male BALB/c mice (CLEA Japan, Inc.) with that same anchor motif as human HLA-A2402.
- GM-CSF Korean Brewery
- IL-4 Gene
- the dendritic cells were pulsed with the peptide of SEQ ID NO: 5 (amino acid initiation position KDR189) and then administered (1x 10 6 cells/mouse) twice to nine BALB/c mice at the right lower quadrant, with an interval of seven days. Seven days later, colon cancer cell line (5x 10 5 cells), Colon 26 (Taiho Pharmaceutical), was inoculated to the right abdomens of the mice, and mice survival and tumor growth were examined.
- KDR expression was analyzed by RT-PCR, as indicated in Fig. 3 , to confirm that KDR was not expressed in Colon 26 cells alone, but expressed in the tumoral tissues of mice inoculated with Colon 26. More specifically, tumor vessels were confirmed to appear in tumoral tissues, and to form the tumor.
- Fig. 3 shows the survival curve of Colon 26-inoculated mice. 35 days after tumor inoculation, two out of nine mice survived when HBSS (Hank's Balanced Salt Solution) was administered alone (100 ⁇ L) as a control; five out of nine mice survived when inoculated with dendritic cells that were not pulsed with peptides; and all of the nine mice survived when inoculated with dendritic cells pulsed with the peptide of SEQ ID NO: 5.
- HBSS Human's Balanced Salt Solution
- Fig. 4 shows the effect on the growth of Colon 26-derived tumors in mice. Compared to the control, suppressive effects on tumor growth were observed when inoculating dendritic cells, and remarkable suppression effects were observed when inoculating dendritic cells pulsed with the peptide of SEQ ID NO: 5.
- the 9-mer peptide (KDR169) of SEQ ID NO: 8 capable of inducing strong CTL activity in Example 4 had only one sequence containing two mismatches (77.8% homology), and two sequences containing three mismatches (66.7% homology).
- Bone marrow was collected from six- to eight-week old male A2/Kb transgenic mice (TGM) expressing human HLA-A0201.
- TGM GM-CSF
- IL-4 Gene
- the dendritic cells were pulsed with the peptide of SEQ ID NO: 30 (amino acid initiation position KDR773), and administered (1x 10 6 cells/mouse) twice to the left lower quadrant of twelve A2/Kb transgenic mice, with an interval of seven days.
- B16 melanoma cells ATCC were inoculated (1x 10 6 cells) to the right abdomen of the mice, and mice survival and tumor growth were examined.
- Fig. 5 shows the survival rate of B16-inoculated mice. 35 days after tumor inoculation, eight of twelve mice survived when HBSS was administered alone (100 ⁇ L) as a control; eleven of twelve mice survived when inoculated with dendritic cells that were not pulsed with peptides; and all twelve mice survived when inoculated with dendritic cells pulsed with the peptide of SEQ ID NO: 30.
- Fig. 6 shows the effect on the growth of B 16 mice-derived tumors in mice. Compared to the control, suppressive effects were observed against tumor growth when inoculating with dendritic cells, and remarkable suppressive effects were observed on inoculating dendritic cells pulsed with the peptide of SEQ ID NO: 30.
- KDR (see, J. Biol. Chem. 1994 Oct 28; 269(43):26988-95 ) was forcedly expressed in HLA-A24-positive human colon cancer HT29 strain (ATCC), according to the method described by Miyake, S. et al., Proc. Natl. Acad. Sci. USA. 93, 1320-1324 (1996 ). More specifically, based on the titer determined by plaque assay of human 293 cells, cells were infected at a specific multiplicity. HT29 cells were plated at a cell density of 10 5 cells in a 60-mm dish, and incubated for 24 hours. On the following day, the medium was removed and exchanged with 200 ⁇ L of viruses continuously diluted with fresh medium.
- HLA-A24-positive HT29 strain forced by adenoviruses to express EGFP (see, Leukemia 1999 Apr; 13(4):605-13 ), was used as a control.
- Cytotoxic activity was measured by 51 Cr release assay for four hours.
- the target cells were cultured in a 6-well plate at a concentration of 1x 10 6 cells/well for 24 hours, infected with adenovirus to which KDR or EGFP has been inserted (Ad-KDR or Ad-EGFP) at MOI 50, and used after 48 hours.
- the target cells were labeled with 100 ⁇ Ci of Na 2 51 CrO 4 at 37°C for one hour, and washed three times with RPMI1640.
- the target cells (1x 10 4 cells/100 ⁇ L) and 100 ⁇ L of effector cells at various concentrations were added (200 ⁇ L in total) to a U-shaped 96-well microtiter plate (Corning), and cultured in a CO 2 incubator at 37°C for four hours. After culturing, 100 ⁇ L of supernatant was collected from each well and measured using a ⁇ counter to calculate cytotoxicity, in the same manner as in Example 2.
- CTL clone C29-169 showed remarkably high cytotoxic effect towards HLA-A24-positive HT29 forcedly expressing KDR, compared to that towards cells forcedly expressing EGFP.
- Cytotoxic activity was measured by a four hour 51 Cr release assay.
- the target cells were labeled with 100 ⁇ Ci of Na 2 51 CrO 4 at 37°C for one hour, then washed three times with RPMI1640.
- Target cells (1x 10 4 cells/100 ⁇ L) and 100 ⁇ L of effector cells at various concentrations were added (200 ⁇ L in total) to a U-shaped 96-well microtiter plate (Corning), and cultured in a CO 2 incubator at 37°C for four hours. After culturing, 100 ⁇ L of supernatant was collected from each well and measured using a ⁇ counter to calculate the cytotoxicity in the same manner as in Example 2.
- CTL clone C29-169 showed cytotoxic effect against HLA-A24-positive HUVEC endogenously expressing KDR, and showed weak cytotoxic effect against HLA-A24-negative HUVEC.
- KDR773-2L peptide (SEQ ID NO: 54) in which the second amino acid from the N terminal in the peptide of SEQ ID NO: 30 (KDR773) has been converted to leucine, the peptide of SEQ ID NO: 29 (KDR775), the peptide of SEQ ID NO: 34 (KDR1328), and the peptide of SEQ ID NO: 33 (KDR772), which bind to HLA-A0201 and which were determined to be effective according to the ELISPOT analysis in Example 7, two types (KWC14-190 and KWC65-190), four types (KWC44-773-2L, KWC76-773-2L, KWC129-773-2L, and KWC134-773-2L), two types (KWC81-775 and KWC85-775), twelve types (KWC16, KWC21, KWC22, KWC47, KWC51, KWC108, KWC117, KWC
- the peptides of SEQ ID NOs: 29, 33, 34, and 40, as well as the peptide of SEQ ID NO: 54, in which the second amino acid from the N terminus in the peptide of SEQ ID NO: 30 is leucine can all induce CTLs having remarkable cytotoxicity, and were thus shown to be effective as epitope peptides.
- the peptide of SEQ ID NO: 29 (amino acid initiation position KDR775) was added to HLA-A0201-positive T2 strain to produce a target cell.
- the cytotoxic effect of the CTL clone (KDR C85-775 (KWC85-775)) obtained from the peptide of SEQ ID NO: 29 in Example 13 was examined by chromium release assay.
- CTL clone C85-775 (KWC85-775) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 29. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 29.
- CTL clone KDR C85-775 (KWC85-775) showed remarkably high cytotoxic effect against HL.4-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide.
- the peptide of SEQ ID NO: 34 (amino acid initiation position KDR1328) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)) obtained from the peptide of SEQ ID NO: 34 in Example 13 was examined by chromium release assay.
- CTL clone KDR C51-1328 (KWC51) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 34.
- absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 34.
- CTL clone KDR C51-1328 showed remarkably high cytotoxicity against HLA-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide.
- the peptide of SEQ ID NO: 54 (KDR773-2L), in which the second peptide of SEQ ID NO: 30 is converted to leucine, was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KDR C44-773-2L (KWC44-773-2L)) obtained from the peptide of SEQ ID NO: 34 in Example 13 was examined by chromium release assay. Fig. 13 shows the results.
- CTL clone KDR C44-773-2L (KWC44-773-2L) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting KDR773-2L. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting KDR773-2L.
- CTL clone KDR C44-773-2L (KWC44-773-2L) showed remarkably high cytotoxic effect against HLA-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide.
- the present inventors determined whether various antibodies can block the cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)), obtained from the peptide of SEQ ID NO: 34, against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR.
- KDR C51-1328 KWC51
- cytotoxic function was inhibited by anti-HLA Class I antibody against HLA antigens displayed by target cells, and anti-CD8 antibody against CD8 used as a marker for cytotoxic T cells.
- mice The actual angiogenesis-inhibiting activity of peptide epitopes was evaluated in mice using a DAS assay (see, Clinical Cancer Research, Vol. 5, 2186-2191, August 1999 ; Cancer Research 62, 6116-6123, November 1, 2002 ).
- a membrane filter was fixed to both sides of a chamber ring, and sterilized with ethylene oxide gas.
- B16 melanoma cells were prepared to 5x 10 5 cells/0.15 mL, and injected into the chamber.
- Mice A2/kb transgenic mice (derived from B 16)) were immobilized face down, and an air sac was produced by injecting 5 to 10 mL of air in to the murine dorsal hypoderm using a syringe. An approximately 1-cm incision was made to the lower part of the sac.
- the chamber ring, filled with cell suspension solution, was implanted in the dorsal hypoderm, and the skin was closed using a skin stapler.
- the mouse skin was decorticated together with the chamber, fixed in an extended form, and marked with a black rubber ring to observe the newly formed blood vessels using a microscope.
- Blood vessels that are newly formed by angiogenic factors released from malignant tumor cells characteristically run in a zigzag manner, and are morphologically different from naturally occurring background blood vessels. Therefore, blood vessels of 3-mm or longer within the rubber ring and running in a zigzag manner were determined to be newly formed blood vessels, and were evaluated using an angiogenesis index (AI). AIs was evaluated into six levels, from 0 to 5 according to the number of tortuous blood vessels. All those with five or more tortuous blood vessels were determined to be 5.
- AI angiogenesis index
- Vaccination was performed 14 days prior to chamber implantation, and repeated after one week (a total of two times) by respectively injecting 5x 10 4 cells of HBSS, DC alone, or peptide-pulsed DC, to the tail vein.
- the results are shown in Fig. 17 and in Table 8, as shown below.
- Fig. 17 and Table 8 showed that the formation of tortuous blood vessels was clearly suppressed in the groups vaccinated with DCs pulsed with the peptides of SEQ ID NO: 40 (amino acid initiation position KDR190), SEQ ID NO: 33 (KDR772), SEQ ID NO: 30 (KDR773), SEQ ID NO: 54 (KDR773-2L), SEQ ID NO: 29 (KDR775), and SEQ ID NO: 46 (KDR1084), respectively, as compared to the group to which DC was administered alone. This indicated a statistically significant angiogenesis inhibitory effect.
- the peptide of SEQ ID NO: 40 (amino acid initiation position KDR190) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KWC65-190) obtained from the peptide of SEQ ID NO: 40 in Example 13 was examined using chromium release assay.
- CTL clone KWC65-190 clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 40.
- absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 40.
- the peptide of SEQ ID NO: 33 (amino acid initiation position KDR772) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KWC72-772) obtained from the peptide of SEQ ID NO: 33 in Example 13 was examined using a chromium release assay.
- CTL clone KWC72-772 clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 33. In contrast, absolutely no cytotoxic effect was observed against control cells that did not present the peptide of SEQ ID NO: 33.
- CTL clone KWC72-772 showed remarkably high cytotoxic effect against HLA-A0201-positive cells presenting KDR peptides on their surface, as compared to activity against cells presenting the EGFP peptide.
- the peptides of SEQ ID NO: 2 (amino acid initiation position KDR1318), SEQ ID NO: 3 (KDR220), SEQ ID NO: 5 (KDR189), and SEQ ID NO: 11 (KDR826) were added to HLA-A24-positive human B-lymphocyte strain A24-LCL (HLA-A24/24, Takara Shuzo) to produce target cells.
- HLA-A24-positive human B-lymphocyte strain A24-LCL HLA-A24/24, Takara Shuzo
- Example 11 the cytotoxic effects of the CTL clones, C7-1318, KWC46, C18-189, and C65-826, described in Example 3, against human colon cancer HT29 strain (ATCC), forced by adenoviruses to express KDR, were examined by chromium release assay. HLA-A24-positive HT29 strain forcedly expressing EGFP was used as a control.
- Example 22 the angiogenesis inhibitory activity of peptide epitopes against the angiogenic response induced by Colon 26 cells in BALB/c mice was evaluated by DAS assay.
- peptide-specific CTLs were detected in PBMC.
- PBMC collected from patients (1x 10 5 cells) were incubated with 10 ⁇ M of each peptide in a U-shaped bottom 96-well plate comprising 200 ⁇ L medium.
- the medium consisted of 45% RPMI1640 medium, 45% AIM-V medium, 10% FBS, 100 U/mL interleukin-2 (IL-2), and 0.1 ⁇ M MEM non-essential amino acid solution. Every three days, half of the medium was removed and replaced with fresh medium containing the corresponding peptide (20 ⁇ M).
- CTL precursor is produced in HLA-A2- and HLA-A24-type cancer patients, as in a healthy donor (Table 9).
- patients A, B, and C are HLA-A24-type patients; and patient D is an HLA-A02-type patient.
- Patients A and D are colon cancer patients; and patients B and C are melanoma patients.
- CMVs refer to the cytomegalovirus-derived peptides used as positive controls.
- the peptides with high A-24 binding affinity are described in Kuzushima, K. et al., Blood 2001, 98(6):p1872-1881 , and such; and the peptides with high A-2 binding affinity are described in Solache, A. et al., J.
- Example 14 As for Example 14 and the like, a standard 51 Cr release assay was performed for four hours to establish CTL lines from the CTL precursors ( Fig. 32 ).
- the present invention provides novel peptides, which induce cytotoxic T cells by targeting the endothelial cells formed in a wide range of tumoral tissues, and which are extremely effective as cancer vaccines.
- the present invention also provides pharmaceuticals for treating and preventing tumors, where the pharmaceuticals comprise these peptides.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Diabetes (AREA)
- Vascular Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Hematology (AREA)
- Cardiology (AREA)
- Dermatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Obesity (AREA)
- Rheumatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Ophthalmology & Optometry (AREA)
- Pain & Pain Management (AREA)
- Emergency Medicine (AREA)
Abstract
Description
- The present invention relates to novel peptides that are extremely useful as cancer vaccines, and pharmaceuticals that comprise these peptides, for treating and preventing tumors.
- Tumor-rejection antigen genes have mainly been identified for malignant melanomas, and accordingly, cancer immunotherapies utilizing these genes are developed. Specifically, with recognition of the importance of CD8-positive T cells in anti-tumor immune responses, cancer vaccine therapy that induces tumor-specific CD8-positive T cells in vivo has received attention, and is being applied to various clinical applications. In addition, the mechanism by which peptides consisting of approximately ten amino acid residues activate T cells via the Class I pathway, by,the assistance of various costimulatory molecules, to induce tumor-specific cytotoxic T cells (CTLs) was also elucidated. Furthermore, peptides restricted to individual HLA molecules are being actively identified.
- However, complete control of tumors is currently impossible. This may be due to tumor cell heterogeneity, a reduction or disappearance of MHC-Class I expression in tumor cells, and the absence of target molecules in the tumor cells. Furthermore, the currently identified tumor antigen peptides exist in some types of tumors, but can not exist in all tumor types. Thus, to resolve these problems, the present inventors did not use tumor cells as target cells, but rather focused on the endothelial cells of tumor vessels. More specifically, endothelial cells have hardly any problems involving a decrease or disappearance of MHC-Class I expression, or heterogeneity. Thus, if CTLs that target tumor vessels can be induced, problems in conventional cancer vaccine therapy, such as the disappearance of Class I and absence of a target molecule, may be overcome, regardless of the type of tumor, and excellent therapeutic effects can be anticipated. Studies on tumor angiogenesis were started from a pioneering hypothesis proposed by Folkman et al, in the 1970's, and have been conducted from various angles. Many studies have been carried out on VEGF-VEGF receptors (VEGFRs) to evaluate their significance in tumor angiogenesis. Angiogenesis inhibitors have been vigorously developed as target-oriented drugs, particularly in cancer therapy, and are already being clinically tested. However, therapies that use this concept for cancer vaccine treatments are not yet in use. One of the reasons may be immunotolerance to VEGFR, which is expressed in normal cells. However, in the 1990's, Plate, Millauer, and Risau et al confirmed that VEGFRs were strongly expressed in the endothelial cells of tumoral tissues. In addition, the immune response to autoantigens such as CEA and HER/neu, which are also expressed in normal cells, is not necessarily one of immunotolerance. Thus, the present inventors reasoned that VEGFRs may be used as targets for cancer vaccine therapy.
- Recently, it was reported that active immunization against VEGFRs can inhibit angiogenesis in tumors, and metastasis (The Journal of Experimental Medicine, 2002, 195:12, 1575-1584). However, this literature merely used soluble VEGFR proteins, and made no investigation of the amino acid sequences of effective peptides.
- The present inventors focused on possible cancer vaccine therapies that target VEGFR2 (KDR/flk-1; referred to below as KDR), where VEGFR2 is strongly expressed in tumoral tissue endothelial cells, and is thought be involved in the proliferation of endothelial cells on the VEGF signal. Furthermore, the present inventors screened for peptides that can be effectively used as vaccines, examining their specificity, and completing this invention.
- More specifically, the present invention provides [1] to [22] as follows.
- [1] A nonapeptide selected from peptides comprising the amino acid sequence of (SEQ ID NO: 2, 3, 5, 8, 11, or 12.
- [2] A peptide with cytotoxic T cell inducibility, wherein one, two, or more amino acids are substituted or added to the amino acid sequence of SEQ ID NO: 2, 3, 5, 8, 11, or 12.
- [3] The peptide of [2], wherein the second amino acid from the N terminus is phenylalanine, tyrosine, methionine, or tryptophan.
- [4] The peptide of [2] or [3], wherein the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, or methionine.
- [5] A nonapeptide or decapeptide selected from peptides comprising the amino acid sequence of SEQ ID NO: 29, 30, 33, 34, 40, or 46.
- [6] A peptide with cytotoxic T cell inducibility, wherein one, two, or more amino acids are substituted or added to the amino acid sequence of SEQ ID NO: 29, 30, 33, 34, 40, or 46.
- [7] The peptide of [6], wherein the second amino acid from the N terminus is leucine or methionine.
- [8] The peptide of [6] or [7], wherein the C-terminal amino acid is valine or leucine.
- [9] A pharmaceutical for treating and/or preventing tumors, wherein the pharmaceutical comprises one or more peptides of any one of [1] to [8].
- [10] A pharmaceutical for treating diabetic retinopathy, chronic rheumatoid arthritis, psoriasis, and atherosclerosis, wherein the pharmaceutical comprises one or more peptides of any one of [1] to [8].
- [11] An exosome that presents on its surface a complex comprising a peptide of any one of [1] to [8], and an HLA antigen.
- [12] The exosome of [11], wherein the HLA antigen is HLA-A24 or HLA-A02.
- [13] The exosome of [12], wherein the HLA antigen is HLA-A2402 or HLA-0201.
- [14] A method for inducing an antigen-presenting cell with high cytotoxic T cell inducibility by using a peptide of any one of [1] to [8].
- [15] A method for inducing a cytotoxic T cell by using a peptide of any one of [1] to [8].
- [16] A method for inducing an antigen-presenting cell with high cytotoxic T cell inducibility, wherein said method comprises the step of introducing a gene that comprises a polynucleotide encoding a peptide of any one of [1] to [8] into an antigen-presenting cell.
- [17] An isolated cytotoxic T cell that is induced by using a peptide of any one of [1] to [8].
- [18] An antigen-presenting cell that presents a complex of an HLA antigen and a peptide of any one of [1] to [8].
- [19] The antigen-presenting cell of [18], which is induced by the method of [14] or [15].
- [20] A vaccine for inhibiting angiogenesis at a diseased site, wherein the vaccine comprises a peptide of any one of [1] to [8] as an active ingredient.
- [21] The vaccine of [20], which is used for administration to a subject whose HLA antigen is HLA-A24 or HLA-A02.
- [22] The vaccine of [20] or [21], which is used to suppress the growth and/or metastasis of malignant tumors.
- The present specification comprises the contents described in the specifications and/or drawings of Japanese Patent Application Nos.
2002-267285 2003-062003 2003-167042 -
-
Fig. 1 shows the cytotoxicity of CTL clones against the target cells. -
Fig. 2 shows the results of HLA-tetramer analyses of established CTL clones:- (a) CTL clones induced by the peptides of this invention, and feeder cells; and
- (b) feeder cells (control).
-
Fig. 3 shows the effect of vaccination using the peptides of this invention on the survival rate of Colon 26-inoculated BALB/c mice. -
Fig. 4 shows the effect of vaccination using the peptides of this invention on the growth of Colon 26-derived tumors. -
Fig. 5 shows the effect of vaccination using the peptides of this invention on the survival rate of B16-inoculated mice. -
Fig. 6 shows the effect of vaccination using the peptides of this invention on the growth of B16 melanoma-derived tumors. Before challenge with a subcutaneous injection of B16 tumor cells, A2/Kb TGM were vaccinated twice, with a one-week interval, with DC pulsed with KDR773 (black circles), DC alone (white triangles), or HBSS (white squares). The figure shows mean tumor growth (P<0.01). -
Fig. 7 shows the cytotoxic effect of CTL clone C29-169 on HLA-A24-positive cells presenting peptides of this invention. -
Fig. 8 shows the cytotoxic effect of CTL clone C29-169 on HLA-A24-positive cells presenting peptides of this invention. -
Fig. 9 shows the cytotoxic effect of CTL clone KDR C85-775 (KWC85-775) on HLA-A0201-positive cells presenting peptides of this invention. -
Fig. 10 shows the cytotoxic effect of CTL clone KDR C85-775 (KWC85-775) on HLA-A0201-positive cells presenting KDR peptides. -
Fig. 11 shows the cytotoxic effect of CTL clone KDR C51-132 (KWC51) on HLA-A0201-positive cells presenting peptides of this invention. -
Fig. 12 shows the cytotoxic effect of CTL clone KDR C51-1328 (KWC51) on HLA-A0201-positive cells presenting KDR peptides. -
Fig. 13 shows the cytotoxic effect of CTL clone KDR C44-773-2L (KWC44-773-2L) on HLA-A0201-positive cells presenting peptides of this invention. -
Fig. 14 shows the cytotoxic effect of CTL clone KDR C44-773-2L (KWC44-773-2L) on HLA-A0201-positive cells presenting peptides of this invention. -
Fig. 15 shows the cytotoxic effect of CTL clone KDR C44-773-2L (KWC44-773-2L) on HLA-A0201-positive cells presenting KDR peptides. -
Fig. 16 shows the inhibitory effect of each type of antibody on the cytotoxic effect of the CTL clones. -
Fig. 17 shows the inhibitory activity of the peptides of this invention on the B 16 cell-induced angiogenic response in A2/Kb TGM. Mice were vaccinated twice with HBSS, unpulsed DC, or DC pulsed with the epitope peptides of this invention (KDR190, KDR772, KDR773, KDR773-2L, KDR775, or KDR1084). The arrows indicate newly formed blood vessels, which run in a characteristic zigzag pattern. -
Fig. 18 shows the suppressive effect of the peptides of this invention on the increase in volume of human tumors in transgenic mice. -
Fig. 19 shows the cytotoxic effect of CTL clone KWC65-190 on HLA-A0201-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 20 shows the cytotoxic effect of CTL clone KWC72-772 on HLA-A0201-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 21 shows the cytotoxic effect of CTL clone KWC72-772 on HLA-A0201-positive cells presenting KDR peptides. HePG2-VEGFR2: black boxes; HePG2-EGFP: white boxes. -
Fig. 22 shows the cytotoxic effect of CTL clone C7-1318 on HLA-A24-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 23 shows the cytotoxic effect of CTL clone KWC46 on HLA-A24-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 24 shows the cytotoxic effect of CTL clone C18-189 on HLA-A24-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 25 shows the cytotoxic effect of CTL clone C65-826 on HLA-A24-positive cells presenting peptides of this invention, in the presence (black boxes) or absence (white boxes) of the peptides. -
Fig. 26 shows the cytotoxic effect of CTL clone C7-1318 on HLA-A24-positive cells presenting peptides of this invention. HT29-VEGFR2: black boxes; HT29-EGFP: white boxes. -
Fig. 27 shows the cytotoxic effect of CTL clone KWC46 on HLA-A24-positive cells presenting peptides of this invention. HT29-VEGFR2: black boxes; HT29-EGFP: white boxes. -
Fig. 28 shows the cytotoxic effect of CTL clone C18-189 on HLA-A24-positive cells presenting peptides of this invention. HT29-VEGFR2: black boxes; HT29-EGFP: white boxes. -
Fig. 29 shows the cytotoxic effect of CTL clone C65-826 on HLA-A24-positive cells presenting peptides of this invention. HT29-VEGFR2: black boxes; HT29-EGFP: white boxes. -
Fig. 30 shows the inhibitory activity of the peptides of this invention on theColon 26 cell-induced angiogenic response in BALB/c mice. The mice were vaccinated twice with HBSS, unpulsed DC, or DC pulsed with an epitope peptide of this invention (KDR189 or KDR826). The arrows indicate newly formed blood vessels, which run in a characteristic zigzag pattern. -
Fig. 31 shows the inhibition ofColon 26 cell-induced angiogenic response in BALB/c by the peptides of this invention. The bars indicate the average, and the vertical error bars indicate standard error. -
Fig. 32 shows the cytotoxic effect of CTL, derived from patients stimulated with the peptides of this invention, on HLA-A24-positive cells, in the presence (black diamonds) or absence (white squares) of the peptides.- (A) The cytotoxic effect of colon cancer patient-derived CTL obtained by stimulation with SEQ ID NO: 8 (in which the amino acid initiation position is KDR169);
- (B) The cytotoxic effect of colon cancer patient-derived CTL obtained by stimulation with SEQ ID NO: 5 (in which the amino acid initiation position is KDR189); and
- (C) The cytotoxic effect of colon cancer patient-derived CTL obtained by stimulation with SEQ ID NO: 3 (in which the amino acid initiation position is KDR220).
- The present inventors first considered that various proteins are presented in vivo on antigen presenting cells after being degraded to 9-mer peptides (nonapeptides), and examined the binding affinity of 9-mer or 10-mer partial peptides of KDR proteins to HLA antigens, which are human major histocompatibiliy antigens (MHC antigens). The lower case letters in the peptide sequences shown on the right of Table 1 indicate the fifth amino acids.
- The amino acid sequence of human KDR protein is well known, disclosed by
US Patent No. 5,861,301 for example, and can be easily obtained by one skilled in the art. The 9-mer and 10-mer peptides can be obtained by synthesizing peptides initiated from any position, based on the full-length amino acid sequence of the obtained KDR protein. Peptides can be synthesized according to conventional methods used in peptide chemistry. Commonly used synthesis methods are described, for example, in literatures such as Peptide Synthesis, Interscience, New York, 1966; The Proteins, vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis (Peputido Gousei), Maruzen Co., Ltd., 1975; Foundations and Experiments in Peptide Synthesis (Peputido Gousei no Kiso to Jikken), Maruzen Co., Ltd., 1985; and Development of Pharmaceuticals, New Series (Iyakuhin no Kaihatsu, Zoku), , and in publications such as International Publication No.WO 99/67288 - Alternatively, software programs now available on the Internet, such as those described in Parker K. C., J. Immunol. 152, 1994, may be used to calculate the binding affinities between various peptides and HLA antigens in silico. Binding affinity with HLA antigens can be measured as described, for example, in Parker, K. C., J. Immunol., 152, 1994; and Nukaya, I., Int. J. Cancer, 80, 1999.
- To obtain sufficient results, A-24 type and A-02 type antigens, said to be highly expressed among the Japanese, are preferably used as the HLA antigens. More preferably, subtypes such as A-2402 and A-0201 are used. However, clinically, by predetermining the HLA antigen type of a patient in need of treatment, a peptide can be appropriately selected to have a high level of binding affinity to that antigen, or level of cytotoxic T cell (CTL) inducibility upon antigen presentation. Furthermore, to obtain peptides with high levels of binding affinity and CTL inducibility, substitution or addition of one, two, or several amino acids may be performed based on the amino acid sequence of the naturally occurring KDR partial peptide. Herein, the term "several" means five or less, or preferably three or less. In addition to peptides in nature, the sequence regularity of the peptides displayed by binding to HLA antigens is already known (J. Immunol., 152, 3913, 1994; Immunogenetics. 41:178, 1995; J. Immunol. 155:4307, 1994). Thus, modifications based on this regularity can also be performed on the obtained peptides. For example, peptides in which the second amino acid from the N terminus is substituted with phenylalanine, tyrosine, methionine, or tryptophan, and in which the C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine may also be preferably used as peptides with high HLA-24 binding affinity. On the other hand, peptides in which the second amino acid from the N terminus is substituted with leucine or methionine, and in which C-terminal amino acid is substituted with valine or leucine may be preferably used as peptides with high HLA-0201 binding affinity. Furthermore, one to two amino acids may also be added to the N and/or C terminus of the peptides.
- However, when a peptide sequence is identical to a portion of an amino acid sequence of an endogenous or exogenous protein with a different function, it may cause side effects such as autoimmune diseases or allergic symptoms against specific substances. Therefore, it is preferable to use available databases to carry out homology searches, to avoid situations in which a sequence matches the amino acid sequence of another protein. Furthermore, if homology searches reveal that no peptides comprising even a one- or two- amino acid difference exist, there is no risk of such problems caused by modifications of the above-mentioned amino acid sequences for increasing the binding affinity with HLA antigens and/or the CTL inducibility.
- As described above, peptides with high binding affinity to the HLA antigens are expected to be highly effective as cancer vaccines. However, it is necessary to determine whether the candidate peptides, which are selected using high binding affinity as an indicator, actually have CTL inducibility or not. Confirmation of CTL inducibility is carried out by inducing antigen-presenting cells comprising human MHC antigens (such as B-lymphocytes, macrophages, and dendritic cells), more specifically dendritic cells derived from human peripheral blood mononuclear leukocytes; stimulating the cells with the peptides; mixing the cells with CD8-positive cells; and then measuring cytotoxicity against the target cells. As the reaction system, transgenic animals generated to express a human HLA antigen may be used (for example, those described in Hum. Immunol. 2000 Aug.; 61(8):764-79 Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response., BenMohamed L., Krishnan R., Longmate J., Auge C., Low L., Primus J., and Diamond DJ.). For example, by radiolabeling the target cells with 51Cr or such, cytotoxicity can be calculated from the radioactivity released from the target cells. Alternatively, the activity can be examined by measuring the IFN-γ produced and released by CTL in the presence of antigen-presenting cells that have immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN-γ monoclonal antibodies.
- The results of examining the CTL inducibility of peptides as described above revealed that those with high binding affinity to HLA antigens do not necessarily have high inducibility. Furthermore, nonapeptides selected from peptides comprising the amino acid sequences of VYSSEEAEL (SEQ ID NO: 2), GYRIYDVVL (SEQ ID NO: 3), SYMISYAGM (SEQ ID NO: 5), RFVPDGNRI (SEQ ID NO: 8), KWEFPRDRL (SEQ ID NO: 11), or DFLTLEHLI (SEQ ID NO: 12), and nonapeptides and decapeptides selected from peptides comprising the amino acid sequence of AMFFWLLLV (SEQ ID NO: 29), VIAMFFWLL (SEQ ID NO: 30), AVIAMFFWL (SEQ ID NO: 33), KLIEIGVQT (SEQ ID NO: 34), YMISYAGMV (SEQ ID NO: 40), or IQSDVWSFGV (SEQ ID NO: 46), showed particularly high CTL inducibility.
- The present invention further provides peptides with cytotoxic T cell inducibility, wherein one, two, or several amino acids are substituted with or added to the amino acid sequence of SEQ ID NO: 2, 3, 6, 8, 11, or 12. Amino acid sequences consisting of nine amino acids of SEQ ID NOs: 2, 3, 5, 8, 11, and 12, in which one, two, or several amino acids are substituted or added, may have CTL inducibility, as long as they do not match the amino acid sequence of another protein. In particular, for example, the second amino acid from the N terminus is preferably substituted to phenylalanine, tyrosine, methionine, or tryptophan, or the C-terminal amino acid is preferably substituted to phenylalanine, leucine, isoleucine, tryptophan, or methionine; or one or two amino acids are added to the N terminus and/or C terminus.
- The present invention also provides peptides with cytotoxic T cell inducibility, wherein one, two, or several amino acids are substituted or added to the amino acid sequence of SEQ ID NOs: 29, 30, 33, 34, 40, or 46. Amino acid sequences consisting of nine or ten amino acids of SEQ ID NOs: 29, 30, 33, 34, 40, and 46, in which one, two, or several amino acids are substituted or added, may have CTL inducibility as long as they do not match the amino acid sequence of another protein. In particular, for example, the second amino acid from the N terminus is preferably substituted to leucine or methionine, or the C-terminal amino acid is preferably substituted to valine or leucine; or one or two amino acids are added to the N terminus and/or C terminus. An example of such a modified peptide is the peptide of SEQ ID NO: 30, in which the second amino acid from the N terminus is substituted to leucine (SEQ ID NO: 54), but this example is not limiting. CTL clones obtained on stimulation with these modified peptides can recognize the original peptides, and cause damage.
- The peptides of this invention can be used alone or in combinations of two or more, as cancer vaccines capable of inducing CTL in vivo. By administering the peptides of this invention, the peptides are presented at a high density on the HLA antigens of antigen-presenting cells. CTLs that specifically react with the complexes formed between the displayed peptides and the HLA antigens are induced, increasing aggression against vascular endothelial cells in the tumor cells to be targeted. Alternatively, antigen-presenting cells on whose cell surface the peptides of this invention are immobilized can be obtained by deriving dendritic cells from a subject, and stimulating them with the peptides of this invention. The obtained antigen-presenting cells are re-administered to the subjects to induce CTL in the subjects. As a result, aggression towards the target cells can be increased.
- More specifically, the present invention provides pharmaceuticals for treating tumors or preventing proliferation, metastasis, and such of tumors, where the pharmaceuticals comprise one or more peptides of this invention. Angiogenesis at pathologic sites is closely associated with not only tumors, but also with diseases such as diabetic retinopathy, chronic rheumatoid arthritis, psoriasis, and atherosclerosis, and the metastasis of solid tumors (Folkman, J., Nature Med. 1:27-31 (1995); Bicknell, R., Harris, A.L., Curr. Opin. Oncol. 8:60-65 (1996)). Therefore, the peptides of this invention can be used to treat tumors, diseases such as diabetic retinopathy, chronic rheumatoid arthritis, psoriasis, atherosclerosis, and metastasis of solid tumors.
- The peptides of this invention were confirmed to inhibit the formation of tortuous blood vessels, which are morphologically different from normal blood vessels, and which are formed in malignant tumoral tissues. The results of analyzing wound healing and fertility in vaccinated mice also confirmed that the peptides do not have an adverse effect on normal physiological angiogenesis. Furthermore, by using CTL clones that recognize the peptides of this invention, cytotoxicity against non-proliferative or proliferative endothelial cells was tested in vitro. These clones showed stronger activity towards proliferative endothelial cells than non-proliferative endothelial cells. More specifically, they can function very specifically for disorders involving proliferative endothelial cells, and particularly cancer.
- In vivo and in vitro stimulation of dendritic cells by the peptides of this invention can be easily performed by exposing the cells to a high concentration of the peptides, which causes these peptides to replace the peptides originally immobilized on the cells. Therefore, the peptides used in this invention must have at least a certain level of binding affinity to HLA antigens.
- The pharmaceuticals of this invention may be directly administered as the peptides of this invention themselves, or may be administered as pharmaceutical compositions that have been formulated by conventional formulation methods. In such cases, the pharmaceuticals of this invention can appropriately include, in addition to the peptides of this invention, carriers, excipients, and such that are ordinarily used for pharmaceuticals, without particular limitations. The pharmaceuticals of this invention can be used for treatment and prevention of various tumors, such as gastric cancer, duodenal cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and brain tumors. The peptides of this invention do not target the tumor cells themselves, but target the endothelial cells of blood vessels that are newly formed in tumoral tissues. Therefore, a wide variety of tumors can be treatment targets, and the pharmaceuticals of this invention are not particularly limited in their use.
- Pharmaceuticals for treating and/or preventing tumors, which comprise a peptide of this invention as an active ingredient, can be administered with adjuvants so that cellular immunity is induced effectively; can be administered with other active ingredients such as antitumor agents; and can be administered in granular forms. Those adjuvants described in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) or such are applicable. Furthermore, the pharmaceuticals of this invention can be administered as liposome formulations, as granular formulations bound to beads of a few micrometers in diameter, and as formulations to which lipids are bound. Administration methods may be carried out, for example, orally, intradermally, or subcutaneously, or through intravenous injection, or such. Systemic administration or local administration to the vicinity of the target tumor may be applicable. Doses of the peptides of this invention can be adjusted appropriately, depending on the disease to be treated, age and weight of the patients, administration methods, and such. Ordinarily, 0.001 mg to 1,000 mg, preferably 0.001 mg to 1,000 mg, more preferably 0.1 mg to 10 mg, of the peptides of this invention are preferably administered once in a few days to a few months.
One skilled in the art can appropriately select suitable doses. - Alternatively, the present invention provides intracellular vesicles which present complexes formed between the peptides of this invention and HLA antigens on their surface. These intracellular vesicles are called exosomes. Exosomes can be prepared, for example, according to the methods specifically described in Published Japanese Translation of International Publication Nos.
Hei 11-510507 2000-512161 - The type of HLA antigens to be used must match that of the subject in need of therapy and/or prophylaxis. For example, for Japanese people, HLA-A24 or HLA-A02, particularly HLA-A2402 or HLA-0201, is often appropriate.
- The present invention also provides methods for inducing antigen-presenting cells using the peptides of this invention. The antigen-presenting cells can be induced by inducing dendritic cells from peripheral blood monocytes; and then contacting (stimulating) them with the peptides of this invention, in vitro or in vivo. Administering the peptides of this invention to subjects induces in the body of the subject antigen-presenting cells to which the peptides of this invention are immobilized. Alternatively, the peptides of this invention can be immobilized to the antigen-presenting cells to be administered to the subject as a vaccine.
- The present invention also provides methods for inducing antigen-presenting cells with a high level of cytotoxic T cell inducibility, wherein said methods comprise the in vitro introduction to an antigen-presenting cell of a gene that comprises a polynucleotide encoding a peptide of this invention. The genes to be introduced may be in the form of DNA or RNA. The introduction methods may be various methods commonly used in the art, such as lipofection, electroporation, and calcium phosphate methods, without particular limitations. More specifically, the methods may be performed as described in, for example. Cancer Res., 56:5672, 1996; J. Immunol., 161:5607, 1998; J. Exp. Med., 184:465, 1996; or Published Japanese Translation of International Publication No.
2000-509281 - Furthermore, the present invention provides methods for inducing CTLs using the peptides of this invention. By administering the peptides of this invention to a subject, CTLs are induced in the body of the subject, enhancing immunity against the angiogenic endothelial cells in the tumoral tissues. Alternatively, the methods may be used for ex vivo therapeutic methods, in which subject-derived antigen-presenting cells, CD8-positive cells, or peripheral blood mononuclear leukocytes are contacted (stimulated) in vitro with the peptides of this invention to induce CTLs, and then the cells are returned to the subject.
- In addition, the present invention provides cytotoxic T cells, that are induced using the peptides of this invention and then isolated. The cytotoxic T cells, which have been induced by stimulation with antigen-presenting cells that present the peptides of this invention, are preferably derived from subjects to be the target of therapy and/or prophylaxis. The cytotoxic T cells can be administered alone or, for the purpose of antitumor effect, in combination with other drugs, including the peptides, exosomes and so on of this invention. The obtained cytotoxic T cells act specifically against target cells presenting the peptides of this invention, or preferably, against target cells presenting the same peptides used for induction. The target cells may be cells that endogenously express KDR, or cells forced to express KDR. Furthermore, cells that present the peptides of this invention on their cell surface due to stimulation by these peptides can also be targeted.
- The present invention also provides antigen-presenting cells that present complexes formed between HLA antigens and the peptides of this invention. The antigen-presenting cells that are obtained by contact with the peptides of this invention, or with nucleotides encoding the peptides of this invention, are preferably derived from subjects to be targeted for therapy and/or prophylaxis. The antigen-presenting cells can be administered as vaccines alone, or in combination with other drugs such as the peptides of this invention, exosomes, and cytotoxic T cells.
- Herein below, the present invention will be specifically described using Examples, but it is not to be construed as being limited thereto.
- Based on the entire amino acid sequence of KDR protein, twelve types of 9-mer peptides (SEQ ID NOs: 1 to 12), and twelve types of 10-mer peptides (SEQ ID NOs: 13 to 24) were predicted in order from that with the highest binding affinity to HLA-A*402, using BioInformatics & Molecular Analysis Section (BIMAS) HLA Peptide Binding Prediction software (http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (Table 1). Table 1 shows the binding affinity of each of the 9 mers and 10 mers in order from the highest value, together with the position of their N termini in the amino acid sequence of the KDR protein. In the table, CE652 (SEQ ID NO: 25) refers to one of the epitopes of tumor antigen CEA (carcinoembryonic antigen) that has been reported by Nukaya, I. (
Int. J. Cancer 80, 1999). HIV peptides (ILKEPVHGV (SEQ ID NO: 55) and RYLRDQQLL (SEQ ID NO: 56)) were used as the negative control peptides. - The peptides were synthesized according to standard solid-phase synthesis methods using Cleaved PepSet (Mimotope, San Diego, CA), and purified by reverse phase HPLC. The purity (>95%) and the type of peptide were individually determined using HPLC and mass spectrometry.
Table 1 Binding of HLA-A*402 to KDR-derived peptides SEQ ID NO: INITIATION POSITION AA SEQUENCE (9mers) BINDING AFFINITY*1 SEQ ID NO: INITIATION POSITION AA SEQUENCE (10mers) BINDING AFFINITY *11 KDR583 WYKLGPQPL 240 13 KDR950 DYVGaIPVDL 420 2 KDR1318 VYSSEEAEL 220 14 KDR434 SYQYgTTQTL 360 3 KDR220 GYRIYDVVL 200 15 KDR1058 DYVRkGDARL 300 4 KDR920 KFGNLSTYL 48 16 KDR304 LYTCaASSGL 200 5 KDR189 SYMISYAGM 38 17 KDR1318 VYSSeEAELL 200 6 KDR828 EFPRDRLKL 33 18 KDR734 LYTCqACSVL 200 7 KDR1152 TFSELVEHL 29 19 KDR926 TYLRsKRNEF 198 8 KDR169 RFVPDGNRI 22 20 KDR353 KYLGyPPPEI 165 9 KDR331 AFGSGMESL 20 21 KDR116 VYVQdYRSPF 150 10 KDR604 HNLDTLWKL 16 22 KDR395 NYTViLTNPI 72 11 KDR826 KWEFPRDRL 12 23 KDR777 FFWLlLVIIL 24 12 KDR998 DFLTLEHLI 9 24 KDR193 SYAGmVFCEA 9.2 25 CE652*2 TYACFVSNL 200 *1 Parker.KC:1994.J.Immunol 152
*2 Nukaya I:1999 - Epstein-Barr virus (EBV)-immortalized B cell lines, TISI (HLA-A24/24) and EHM (HLA-A3/3), were provided by Takara Shuzo Biotechnology Research Laboratory.
- HLA serotyping was performed using 7 mL of peripheral blood collected from a healthy subject. Peripheral blood mononuclear lymphocytes (PBMCs) were isolated from HLA-A24-positive peripheral blood using Ficoll Paque (Pharmacia) specific gravity centrifugation.
- The obtained PBMCs were left to stand for ten hours in a culture flask (Corning, 43072). After removing the suspended cells, the cells that adhered to the flask were cultured in AIM-V medium (Invitrogen) supplemented with 2% autoserum by adding 1,000 U/mL of human GM-CSF (provided by Kirin Brewery), and 1,000 U/mL of human IL-4 (Genzyme). Five days later, the cells were cultured for another 48 hours in 10 µg/mL of OK-432 (provided by Chugai Pharmaceutical Co., Ltd.), and were used as antigen-presenting cells for CTL induction. Dendritic cells (DCs) were confirmed to be DCs by reaction with FITC-labeled anti-ClassII, CD80, and CD86 antibodies, and with PE-labeled anti-ClassI, CD11c, CD40 (all from Beckton-Dickinson), and CD83 (Immunotech) antibodies; followed by analysis of surface antigens by FACS-Calibur (Beckton-Dickinson) using Cell Quest software.
- Pre-induced DCs were pulsed for four hours at 20°C with twelve kinds of 9-mer peptides (SEQ ID NOs: 1 to 12) (20 µg/mL), that were predicted in Example 1 to have high binding affinity, in the presence of 3 µg/mL of β2-microglobulin. The resulting DCs were mixed with CD8-positive cells selected by magnetic beads (Dynabeads M-450 and Detachabeads) from PBMC, at a ratio of 1:20 or 1:2, and then cultured in a 48-well plate (Corning), in the presence of 10 ng/mL of human IL-7 (Genzyme). Three days later, the final concentration of 10 U/mL of IL-2 (Sigma) was added to the culture. And seven and 14 days later, the same DCs were stimulated to induce CTLs, and 20 days later, cytotoxicity was measured for each well using peptide-pulsed TISI cells as targets. Only the positive wells were cultured by a stimulation method with allo-PBMC, EBV-immortalized B cell lines (EHM), and 30 ng/mL of anti-CD3 antibody. CTLs were functionally analyzed 14 days later.
- Cytotoxic activity was evaluated by a four-hour 51Cr release assay. A concentration of 20 µg/mL of the target cells were pulsed with peptides overnight. The target cells were labeled with 100 µCi of Na2 51CrO4 at 37°C for one hour, and then washed three times with RPMI1640. The target cells (1x104/100 µL) and 100 µL of effector cells at various concentrations with a total volume of 200 µL were placed into a U-shaped 96-well microtiter plate (Corning), and cultured at 37°C in a CO2 incubator for four hours. After culturing. 100 µL of supernatant was collected from each well, and measured using a γ counter. Natural decay was the radioactivity of the target cells and the medium, and maximum decay was the radioactivity of the target cells and 1 M HCl. Cytotoxic activity (percentage of specific lysis) was calculated by the following formula:
- Six types of CTL lines were established as a result, as shown in Table 2. In particular, nonapeptides shown by SEQ ID NO: 2 (amino acid initiation position KDR1318), SEQ ID NO: 3 (KDR220), SEQ ID NO: 5 (KDR189), SEQ ID NO: 8 (KDR169), SEQ ID NO: 11 (KDR826), and SEQ ID NO: 12 (KDR998) were shown to be effective as epitope peptides.
Table 2 SEQ ID NO: INITIATION POSITION AA SEQUENCE BINDING*1 AFFINITY CYTOTOXIC ACTIVITY × 20 × 2 Pep(+) Pep(- -) Pep(+) Pep(- -) 1 KDR583 WYKLGPQPL 240 6% 4% 0% 0% 2 KDR1318 VYSSEEAEL 220 39% 21% 8% 3% 3 KDR220 GYRIYDVVL 200 57% 2% 20% 0% 4 KDR920 KFGNLSTYL 48 3% 4% 0% 2% 5 KDR189 SYMISYAGM 38 41% 3% 20% 0% 6 KDR828 EFPRDRLKL 33 3% 2% 1% 0% 7 KDR1152 TFSELVEHL 29 0% 0% 0% 0% 8 KDR169 RFVPDGNRI 22 98% 2% 53% 0% 9 KDR331 AFGSGMESL 20 6% 6% 0% 0% 10 KDR604 KNLDTLWKL 16 3% 2% 1% 1% 11 KDR826 KWEFPRDRL 12 23% 0% 11% 0% 12 KDR998 DFLTLEHLI 9 13% 4% 8% 0% 25 CE652*2 TYACFVSNL 200 29% 16% 7% 1% *1 Parker.KC:1994.J.Immnunol 152
*2 Nukaya I:1999 - The six types of CTL lines established in Example 2 were diluted to 0.3, 1, or 3 cells/well in a U-shaped 96-well plate.
7x 104 cells/well of allo-PBMC,1x 104 cells/well of EHM, 30 ng/mL of anti-CD3 antibody, and 125 U/mL of IL-2 were added to each well, and AIM-V supplemented with 5% autoserum was added thereto, so that total concentration was 150 µL/well. Ten days later, 50 µL/well of IL-2-supplemented medium adjusted was added to a final IL-2 concentration of 125 U/mL. CTLs were functionally analyzed on the 14th day, and CTLs with activity were cultured on a large scale. - As a result, from each of the five types of nonapeptides shown by SEQ ID NO: 8 (amino acid initiation position KDR169), SEQ ID NO: 5 (KDR189), SEQ ID NO: 3 (KDR220), SEQ ID NO: 2 (KDR1318), and SEQ ID NO: 11 (KDR826), eight types (C13-169, C19-169, C29-169, C53-169, C72-169, C61-169, K5-169, and K25-169), two types (C12-189 and C18-189), eleven types (KWC3, 23, 25, 26, 36, 42, 46, 58, 61, 70, and 77), one type (C7-1318), and one type (C65-826) of CTL clones were established, respectively (Table 3).
Table 3 CYTOTOXIC ACTIVITY ×20 ×2 CLONE NAME Pep(+) Pep(- -) Pep(+) Pep(- -) C13-169 98% 2% 66% 2% C19-169 94% 2% 50% 0% C29-169 100% 1% 70% 0% C53-169 100% 2% 91% 2% C72-169 87% 1% 39% 0% C61-169 88% 0% 38% 0% K5-169 36% 0% 6% 0% K25-169 27% 0% 5% 0% C12-189 27% 18% 7% 3% C18-189 67% 18% 28% 7% KWC3 54% 2% 11% 1% KWC23 95% 1% 49% 1% KWC25 93% 1% 70% 1% KWC26 87% 1% 34% 0% KWC36 62% 2% 11% 2% KWC42 67% 0% KWC46 94% 1% 51% 0% KWC58 76% 0% KWC61 70% 2% 19% 1% KWC70 92% 3% 64% 2% KWC77 99% 3% 70% 2% C65-826 38% 6% 8% 3% C7-1318 84% 1% 62% 1% - The degrees of cytotoxicity of the ten types of CTL clones established in Example 3 were examined by varying their ratio with the target cells (E:T ratio).
- As a result, C53-169 derived from the nonapeptide of SEQ ID NO: 8 (amino acid initiation position KDR169) showed the strongest cytotoxicity.
- An HLA-tetramer was synthesized from HLA-A*2402 and the peptide of SEQ ID NO: 8 (amino acid initiation position KDR169). More specifically, plasmid vectors expressing the H chain (approximately 35 kDa) and L chain (approximately 11 kDa) were produced individually, and their recombinant protein was expressed using E. coli. The H-chain plasmid vector consists only of the extracellular domain, in which the carboxyl terminus was substituted with a biotinylation enzyme recognition sequence. The H chain and L chain form an HLA peptide complex by refolding with an antigenic peptide. The HLA peptide complexes were biotinylated by a biotinylation enzyme, excess biotin was removed by gel filtration chromatography and ion exchange chromatography, and the biotinylated HLA peptide complexes were isolated and purified. HLA-tetramer was synthesized by reacting the obtained biotinylated HLA peptide complex with streptavidin (SA) at a mole ratio of 4:1 to form a tetramer. When analyzing the samples, SA fluorescently labeled with phycoerythrin (PE) (SA-PE) was used to form the tetramer. CD8 molecule is known to bind to the 245th amino acid in the α3 domain, which is alanine, with the exception of portions of HLA-A, B, and C. Therefore, the present inventors mutagenized the alanine in the HLA-tetramers to valine, to produce a mutant HLA-tetramer capable of selectively detecting CTLs with high avidity towards HLA peptide complexes, independent of the binding between the CD8 molecule and the α3 domain.
- Established CTL clones were detected by the synthesized HLA-A24 KDR169-tetramer mutant. As a result, as shown in
Fig. 2 , the established CTL clones were found to be strongly stained by the tetramer-positive and CD8-positive fractions, and to specifically recognize complexes formed between the peptides of this invention and HLA antigens. The signals in the tetramer-negative and CD8-negative fractions are due to feeder cells. - In a similar manner to Example 1, 15 types of 9-mer peptides (SEQ ID NOs: 26 to 40), and 12 types of 10-mer peptides (SEQ ID NOs: 41 to 52) were predicted from the entire amino acid sequence of the KDR protein, in order from highest binding affinity with HLA-A*0201, using HLA Peptide Binding Prediction software (http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (Table 4). Table 4 shows the binding affinity of each of the 9 mers and 10 mers in order from the highest value, together with the position of their N terminal in the amino acid sequence of the KDR protein. In the table, CEA588 (SEQ ID NO: 53) refers to one of the epitopes of tumor antigen CEA (carcinoembryonic antigen) reported by Tanaka, H. et al. (poster presentation, AACR #3669, vol. 42, p681-682, March 2001).
Table 4 Binding of HLA-A*0201 to KDR-derived peptides SEQ ID NO: INITIATION POSITION AA SEQUENCE (9mers) BINDING AFFINITY SEQ ID NO: INITIATION POSITION AA SEQUENCE ( 10mers BINDING AFFINITY 26 KDR1093 VLLWEIFSL 1792 41 KDR1094 LLWEIFSLGA 1092 27 KDR 633 SLQDQGDYV 769 42 KDR 5VLLAvALWLC 539 28 KDR 5VLLAVALWL 739 43 KDR 313 LMTKkNSTFV 470 29 KDR 775AMFFWLLLV 427 44 KDR 779 WLLLvIILRT 292 30 KDR 773VIAMFFWLL 270 45 KDR 505 ALIEgKNTKTV 285 31 KDR1034 ILLSEKNVV 179 46 KDR1084 IQSDvWSFGV 285 32 KDR 604 KNLDTLWKL 128 47 KDR 733 GLYTcQACSV 223 33 KDR 772AVIAMFFWL 113 48 KDR 780 LLLViILRTV 201 34 KDR1328 KLIEIGVQT 107 49 KDR 6LLAVaLWLCV 201 35 KDR 698 IMWFKDNET 76 50 KDR 137 YITEnKNKTV 180 36 KDR 608 TLWKLNATM 41 51 KDR1035 LLSEkNVVKI 167 37 KDR 491 FQGGNKIEV 32 52 KDR 4KVLLaVALWL 133 38 KDR 435 YQYGTTQTL 32 39 KDR 505 ALIEGKNKT 31 40 KDR 190 YMISYAGMV 30 53 CEA 588*1 DVLYGPDTPI 0.25 *1 Tanaka H et al.:Poster presented at AACR 2001 - CTLs were induced by a vaccination method, using the 16 types of 9-mer and 10-mer peptides (SEQ ID NO: 26 to 41) predicted in Example 6 on transgenic mice that express human HLA (A0201) (Hum. Immunol. 2000 Aug.;61(8):764-79 Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLAA*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response., BenMohamed L., Krishnan R., Longmate J., Auge C., Low L., Primus J., and Diamond DJ.).
- Six- to eight-week old BALB/C(H-2d) mice were obtained from CLEA Japan. A2/Kb transgenic mice (TGM) were provided by F. Jim Primus, Ph. D. at Vanderbilt-Ingram Cancer Center. T2 cells (TAP-deficient and HLA-A*0201-positive cells) were provided by Prof. H. Shiku at Mie University's Third Department of Internal Medicine.
- Bone marrow was collected from mouse femur and tibia, and lymphocytes and granulocytes were removed using anti-CD4, CD8, Gr-1 antibodies (Beckton-Dickinson), anti-B220 antibody (Bioscience) and rabbit complement (PeL-Freez). The obtained cells were cultured in a 6-well plate. Suspended cells were collected the following day to culture in RPMI1640 (Invitrogen) medium supplemented with 10% FBS in another 6-well plate, to which 1,000 U/mL of mouse GM-CSF (provided by Kirin Brewery) and 1,000 U/mL of mouse IL-4 (PEPRO TECH) were added. Three days later, half of the above-mentioned medium was changed, and another two days later, the cells were cultured for 20 hours in 10 µg/mL of OK-432. The resulting suspended cells were used as DCs were reacted with FITC-labeled anti-ClassII and CD40 antibodies, and PE-labeled anti-ClassI, CD11c, CD80, and CD86 antibodies (all from Beckton-Dickinson), and confirmed to be DCs by analysis of the surface antigens by FACS-Calibur (Beckton-Dickinson) using Cell Quest software.
- Next, 100 µg of each peptide, 140 µg of HbcAg120-140 helper peptide, and 100 µL of IFA were mixed (200 µL in total). The mixture was administered subcutaneously to the right abdomen on
day 0, and to the left abdomen on day 11. The spleens of the vaccinated mice were collected onday 21, erythrocytes were hemolytically removed using red lysis buffer (Sigma), and a portion of those cells were used as responder cells for IFN-γ ELISPOT assay. The remaining cells were placed into a 24-well plate (Corning) at6x 106 cells/well with feeder cells at a ratio of 3:1, and stimulated again. Their cytotoxicity was measured five days later. Erythrocyte-free splenocytes, which were derived from syngeneic mice, were cultured for three days in the presence of 25 µg/mL of lipopolysaccharide (LPS) to use as feeder cells. - In this Example, the IFN-γ ELISPOT method was used to evaluate CTL inducibility according to SPOTs produced by IFN-γ producing cells. Flat-bottomed 96-well multiscreen plate MAHA S45 (Millipore) was treated at 4°C overnight with anti-mouse IFN-γ monoclonal antibody (Pharmingen). On the following day, the plate washed with PBS containing 0.05
% Tween 20, and then reacted with a blocking buffer at room temperature for two hours. Thereafter, peptide-pulsed T2 cells and unpulsed T2 cells were added to each well at 105 cells/100 µL. The splenocytes of vaccinated mice were further added to each well at a maximum of4x 106 cells/100 µL (200 µL in total), and cultured overnight at 37°C. The next day, each well was washed, and reacted for two hours with biotinylated rat anti-mouse IFN-γ antibodies (Pharmingen). After washing the plate thoroughly, Extravidin was added to each well and reacted at room temperature for two hours. After washing, alkaline phosphatase conjugate substrate (BIO-RAD) was added to the wells, which were left to stand at room temperature for five minutes. The blue spots resulting from IFN-γ production were measured using KS ELISPOT compact release (Carl Zeiss). -
-
- As a result, five types of epitope peptides, SEQ ID NO: 29 (amino acid initiation position KDR775), SEQ ID NO: 30 (KDR773), SEQ ID NO: 33 (KDR772), SEQ ID NO: 34 (KDR1328), and SEQ ID NO: 40 (KDR190) were obtained by IFN-γELISPOT assay (Table 5).
Table 5 ELISPOT Analysis SEQ ID NO: INITIATION POSITION AA SEQUENCE BINDING AFFINITY ELISPOT ANALYSIS X 40 (SFC) MOUSE 1MOUSE2 pep(+) pep(- -) pep(+) pep(- -) 26 KDR1093 VLLWEIFSL 1792 5 8 7 1 41 KDR1094 LLWEIFSLGA 1092 0 0 0 0 27 KDR 633 SLQDQGDYV 769 0 0 0 0 28 KDR 5VLLAVALWL 739 6 0 7 3 29 KDR 775AMFFWLLLV 427 95 32 322 17 30 KDR 773VIAMFFWLL 270 171 6 193 1 31 KDR1034 ILLSEKNW 179 0 0 0 0 32 KDR 604 KNLDTLWKL 128 0 7 0 0 33 KDR 772AVIAMFFWL 113 88 19 143 31 34 KDR1328 KLIEIGVQT 107 0 0 81 5 35 KDR 698 IMWFKDNET 76 0 0 0 0 36 KDR 608 TLWKLNATM 41 0 0 0 0 37 KDR 491 FQGGNKIEV 32 0 0 0 0 38 KDR 435 YQYGTTQTL 32 0 0 0 0 39 KDR 505 ALIEGKNKT 31 0 0 0 0 40 KDR 190 YMISYAGMV 30 122 6 33 11 53 CEA 588±1 DVLYGPDTPI 0.25 229 2 230 6 ±1 Tanaka H et al.:Poster presented at AACR 2001 - Bone marrow was collected from six- to eight-week old male BALB/c mice (CLEA Japan, Inc.) with that same anchor motif as human HLA-A2402. As in Example 7, GM-CSF (Kirin Brewery) (1,000 U/mL) and IL-4 (Genzyme) (1,000 U/mL) were added to the bone marrow and cultured to prepare dendritic cells. The dendritic cells were pulsed with the peptide of SEQ ID NO: 5 (amino acid initiation position KDR189) and then administered (
1x 106 cells/mouse) twice to nine BALB/c mice at the right lower quadrant, with an interval of seven days. Seven days later, colon cancer cell line (5x 105 cells), Colon 26 (Taiho Pharmaceutical), was inoculated to the right abdomens of the mice, and mice survival and tumor growth were examined. - First, KDR expression was analyzed by RT-PCR, as indicated in
Fig. 3 , to confirm that KDR was not expressed inColon 26 cells alone, but expressed in the tumoral tissues of mice inoculated withColon 26. More specifically, tumor vessels were confirmed to appear in tumoral tissues, and to form the tumor. -
Fig. 3 shows the survival curve of Colon 26-inoculated mice. 35 days after tumor inoculation, two out of nine mice survived when HBSS (Hank's Balanced Salt Solution) was administered alone (100 µL) as a control; five out of nine mice survived when inoculated with dendritic cells that were not pulsed with peptides; and all of the nine mice survived when inoculated with dendritic cells pulsed with the peptide of SEQ ID NO: 5. -
Fig. 4 shows the effect on the growth of Colon 26-derived tumors in mice. Compared to the control, suppressive effects on tumor growth were observed when inoculating dendritic cells, and remarkable suppression effects were observed when inoculating dendritic cells pulsed with the peptide of SEQ ID NO: 5. - Homology searches for the peptides of this invention (SEQ ID NO: 2 (amino acid initiation position KDR1318), SEQ ID NO: 3 (KDR220), SEQ ID NO: 5 (KDR189), SEQ ID NO: 8 (KDR169), SEQ ID NO: 11 (KDR826), SEQ ID NO: 30 (KDR773), and SEQ ID NO: 40 (KDR190)) were carried out by using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). No peptides with a sequence completely identical to any one of these peptides could be found (Table 6). The 9-mer peptide (KDR169) of SEQ ID NO: 8 capable of inducing strong CTL activity in Example 4 had only one sequence containing two mismatches (77.8% homology), and two sequences containing three mismatches (66.7% homology). The peptide of SEQ ID NO: 5 (KDR189), whose remarkable in vivo anti-tumor effect was observed in Example 8, had only one sequence containing three mismatches (66.7% homology).
Table 6 Homology analysis using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) KDR169 KDR189 KDR826 KDR220 KDR1318 KDR773 KDR190 IDENTITY (9/9) 0 0 0 0 0 0 0 IDENTITY (8/9) 0 0 3 0 0 0 0 IDENTITY (7/9) 1 0 2 0 2 2 0 IDENTITY (6/9) 2 1 - 0 - 2 0 - Bone marrow was collected from six- to eight-week old male A2/Kb transgenic mice (TGM) expressing human HLA-A0201. As in Example 8, GM-CSF (Kirin Brewery) (1,000 U/mL) and IL-4 (Genzyme) (1,000 U/mL) were added to the bone marrow and then cultured to prepare dendritic cells. The dendritic cells were pulsed with the peptide of SEQ ID NO: 30 (amino acid initiation position KDR773), and administered (
1x 106 cells/mouse) twice to the left lower quadrant of twelve A2/Kb transgenic mice, with an interval of seven days. Seven days later, B16 melanoma cells (ATCC) were inoculated (1x 106 cells) to the right abdomen of the mice, and mice survival and tumor growth were examined. -
Fig. 5 shows the survival rate of B16-inoculated mice. 35 days after tumor inoculation, eight of twelve mice survived when HBSS was administered alone (100 µL) as a control; eleven of twelve mice survived when inoculated with dendritic cells that were not pulsed with peptides; and all twelve mice survived when inoculated with dendritic cells pulsed with the peptide of SEQ ID NO: 30. -
Fig. 6 shows the effect on the growth of B 16 mice-derived tumors in mice. Compared to the control, suppressive effects were observed against tumor growth when inoculating with dendritic cells, and remarkable suppressive effects were observed on inoculating dendritic cells pulsed with the peptide of SEQ ID NO: 30. - Using adenoviruses, KDR (see, J. Biol. Chem. 1994 ) was forcedly expressed in HLA-A24-positive human colon cancer HT29 strain (ATCC), according to the method described by Miyake, S. et al., Proc. Natl. Acad. Sci. USA. 93, 1320-1324 (1996). More specifically, based on the titer determined by plaque assay of human 293 cells, cells were infected at a specific multiplicity. HT29 cells were plated at a cell density of 105 cells in a 60-mm dish, and incubated for 24 hours. On the following day, the medium was removed and exchanged with 200 µL of viruses continuously diluted with fresh medium.
- The cells were incubated at 37°C for one hour, and then growth medium was added to the cells, which were cultured for 48 hours to be used as target cells. HLA-A24-positive HT29 strain, forced by adenoviruses to express EGFP (see, Leukemia 1999 Apr; 13(4):605-13), was used as a control. HT29 strains forcedly expressing KDR and EGFP, respectively, can potentially present partial peptides of each protein on their respective cell surface.
- Using CTL clone C29-169, induced from the peptide of SEQ ID NO: 8 (amino acid initiation position KDR169), as described in Example 3, which binds to HLA-A24, the cytotoxic effect on HLA-A24-positive HT29 cells forced to express KDR was measured by chromium release assay. The results are shown in
Fig. 7 . - Cytotoxic activity was measured by 51Cr release assay for four hours. The target cells were cultured in a 6-well plate at a concentration of
1x 106 cells/well for 24 hours, infected with adenovirus to which KDR or EGFP has been inserted (Ad-KDR or Ad-EGFP) atMOI 50, and used after 48 hours. The target cells were labeled with 100 µCi of Na2 51 CrO4 at 37°C for one hour, and washed three times with RPMI1640. The target cells (1x 104 cells/100 µL) and 100 µL of effector cells at various concentrations were added (200 µL in total) to a U-shaped 96-well microtiter plate (Corning), and cultured in a CO2 incubator at 37°C for four hours. After culturing, 100 µL of supernatant was collected from each well and measured using a γ counter to calculate cytotoxicity, in the same manner as in Example 2. - As a result, as shown in
Fig. 7 , CTL clone C29-169 showed remarkably high cytotoxic effect towards HLA-A24-positive HT29 forcedly expressing KDR, compared to that towards cells forcedly expressing EGFP. - The cytotoxic effect of the CTL clone C29-169 described in Example 3, against HUVEC KT5 strain, derived from HLA-A24-positive human umbilical cord vascular endothelial cells that endogenously express KDR, was examined using KDR-positive and HLA-A24-negative HUVEC strain, P8, as a control. These cells present partial peptides of KDR on their cell surface.
- Cytotoxic activity was measured by a four hour 51Cr release assay. The target cells were labeled with 100 µCi of Na2 51 CrO4 at 37°C for one hour, then washed three times with RPMI1640. Target cells (
1x 104 cells/100 µL) and 100 µL of effector cells at various concentrations were added (200 µL in total) to a U-shaped 96-well microtiter plate (Corning), and cultured in a CO2 incubator at 37°C for four hours. After culturing, 100 µL of supernatant was collected from each well and measured using a γ counter to calculate the cytotoxicity in the same manner as in Example 2. - As apparent from the results of
Fig. 8 , CTL clone C29-169 showed cytotoxic effect against HLA-A24-positive HUVEC endogenously expressing KDR, and showed weak cytotoxic effect against HLA-A24-negative HUVEC. - By using the peptide of SEQ ID NO: 40 (amino acid initiation position KDR190), KDR773-2L peptide (SEQ ID NO: 54) in which the second amino acid from the N terminal in the peptide of SEQ ID NO: 30 (KDR773) has been converted to leucine, the peptide of SEQ ID NO: 29 (KDR775), the peptide of SEQ ID NO: 34 (KDR1328), and the peptide of SEQ ID NO: 33 (KDR772), which bind to HLA-A0201 and which were determined to be effective according to the ELISPOT analysis in Example 7, two types (KWC14-190 and KWC65-190), four types (KWC44-773-2L, KWC76-773-2L, KWC129-773-2L, and KWC134-773-2L), two types (KWC81-775 and KWC85-775), twelve types (KWC16, KWC21, KWC22, KWC47, KWC51, KWC108, KWC117, KWC132, KWC151, KWC163, KWC156, and KWC159), and one type (KWC72-772) of CTL clones, respectively, were obtained as in Example 3. The respective cytotoxicities are shown in Table 7.
Table 7 CLONE NAME CYTOTOXIC ACTIVITY × 20 × 2 Pep(+) Pep(- -) Pep(+) Pep(- -) KWC14 -190 43% 0% 9% 0% KWC65 -190 83% 0% 27% 0% kWC44 - 773 -2L 87% 0% 64% 0% KWC76 -773 -2L 83% 2% 68% 1% KWC129 -773 -2L 89% 0% 82% 1% KWC134 -773 -2L 89% 2% 54% 2% KWC81-775 86% 0% 64% 0% hWC85 -775 90% 0% 63% 0% KWC16 95% 0% 85% 0% KWC21 100% 0% 91% 0% KWC22 93% 0% 77% 0% KWC47 109% 2% 87% 1 % KWC51 101% 0% 93% 1% KWC108 71% 2% 23% 2% KWC117 83% 0% 42% 0 % KWC132 102% 0% 55% 0 % KWC151 102% 0% 101% 0% KWC153 47% 0% 18% 0% KWC156 64% 0% 23% 0% KWC159 93% 0% 86% 0% KMC72-772 101% 0% 67% 0% - As apparent from the results in Table 7, the peptides of SEQ ID NOs: 29, 33, 34, and 40, as well as the peptide of SEQ ID NO: 54, in which the second amino acid from the N terminus in the peptide of SEQ ID NO: 30 is leucine, can all induce CTLs having remarkable cytotoxicity, and were thus shown to be effective as epitope peptides.
- The peptide of SEQ ID NO: 29 (amino acid initiation position KDR775) was added to HLA-A0201-positive T2 strain to produce a target cell. By using the cell, the cytotoxic effect of the CTL clone (KDR C85-775 (KWC85-775)) obtained from the peptide of SEQ ID NO: 29 in Example 13 was examined by chromium release assay.
- In the results, shown in
Fig. 9 , CTL clone C85-775 (KWC85-775) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 29. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 29. - The cytotoxic effect of the CTL clone (KDR C85-775 (KWC85-775)) against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR, was examined by chromium release assay. HLA-A0201-positive HePG2 forced to express EGFP was used as control.
- In the results, show in
Fig. 10 , CTL clone KDR C85-775 (KWC85-775) showed remarkably high cytotoxic effect against HL.4-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide. - The peptide of SEQ ID NO: 34 (amino acid initiation position KDR1328) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)) obtained from the peptide of SEQ ID NO: 34 in Example 13 was examined by chromium release assay.
- In the results, show in
Fig. 11 , CTL clone KDR C51-1328 (KWC51) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 34. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 34. - The cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)) against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR, was examined by chromium release assay. HLA-A0201-positive HePG2 forced to expressing EGFP was used as control.
- In the results, shown in
Fig. 12 , CTL clone KDR C51-1328 (KWC51) showed remarkably high cytotoxicity against HLA-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide. - The peptide of SEQ ID NO: 54 (KDR773-2L), in which the second peptide of SEQ ID NO: 30 is converted to leucine, was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KDR C44-773-2L (KWC44-773-2L)) obtained from the peptide of SEQ ID NO: 34 in Example 13 was examined by chromium release assay.
Fig. 13 shows the results. - In the results, shown in
Fig. 13 , CTL clone KDR C44-773-2L (KWC44-773-2L) clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting KDR773-2L. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting KDR773-2L. - The cytotoxic effect of the CTL clone (KDR C44-773-2L (KVJC44-773-2L) obtained from the peptide of SEQ ID NO: 54 (KDR773-2L), in which the second peptide of SEQ ID NO: 30 has been converted to leucine, was examined by chromium release assay using HL4-A0201-positive T2 strain pulsed with the unmodified peptide of SEQ ID NO: 30 (amino acid initiation position KDR773) as the target cell.
- In the results, shown in
Fig. 14 , CTL clones obtained by stimulation with the modified peptide could recognize and damage the post-modification peptide. - The cytotoxic effect of the CTL clone (KDR C44-773-2L (KWC44-773-2L)) against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR, was examined by chromium release assay. HLA-A0201-positive HePG2 forced to express EGFP was used as control.
- In the results, shown in
Fig. 15 , CTL clone KDR C44-773-2L (KWC44-773-2L) showed remarkably high cytotoxic effect against HLA-A0201-positive cells presenting KDR peptides on the surface, compared to activity against cells presenting the EGFP peptide. - The present inventors determined whether various antibodies can block the cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)), obtained from the peptide of SEQ ID NO: 34, against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR.
- In the results, shown in
Fig. 16 , cytotoxic function was inhibited by anti-HLA Class I antibody against HLA antigens displayed by target cells, and anti-CD8 antibody against CD8 used as a marker for cytotoxic T cells. - The actual angiogenesis-inhibiting activity of peptide epitopes was evaluated in mice using a DAS assay (see, Clinical Cancer Research, Vol. 5, 2186-2191, August 1999; Cancer Research 62, 6116-6123, November 1, 2002).
- Specifically, a membrane filter was fixed to both sides of a chamber ring, and sterilized with ethylene oxide gas. B16 melanoma cells were prepared to
5x 105 cells/0.15 mL, and injected into the chamber. Mice (A2/kb transgenic mice (derived from B 16)) were immobilized face down, and an air sac was produced by injecting 5 to 10 mL of air in to the murine dorsal hypoderm using a syringe. An approximately 1-cm incision was made to the lower part of the sac. The chamber ring, filled with cell suspension solution, was implanted in the dorsal hypoderm, and the skin was closed using a skin stapler. Six days after implantation, the mouse skin was decorticated together with the chamber, fixed in an extended form, and marked with a black rubber ring to observe the newly formed blood vessels using a microscope. Blood vessels that are newly formed by angiogenic factors released from malignant tumor cells characteristically run in a zigzag manner, and are morphologically different from naturally occurring background blood vessels. Therefore, blood vessels of 3-mm or longer within the rubber ring and running in a zigzag manner were determined to be newly formed blood vessels, and were evaluated using an angiogenesis index (AI). AIs was evaluated into six levels, from 0 to 5 according to the number of tortuous blood vessels. All those with five or more tortuous blood vessels were determined to be 5. - Vaccination was performed 14 days prior to chamber implantation, and repeated after one week (a total of two times) by respectively injecting
5x 104 cells of HBSS, DC alone, or peptide-pulsed DC, to the tail vein. The results are shown inFig. 17 and in Table 8, as shown below.Table 8 Effect of DAS assay INOCULATION PEPTIDE STIMULATION ANGIOGENESIS INDEX (AI) P VEHICLE - (0.7 ± 0.2) HBSS - (4.5 ± 0.2) DC NONE (4.2 ± 0.4) DC KDR190 (SEQ ID NO:40) (3.6 ± 0.5) 0.3453 DC KDR772 (SEQ ID NO:33) (2.8 ± 0.5) 0.0346 DC KDR773 (SEQ ID NO:30) (2.2 ± 0.7) 0.0034 DC KDR773-2L (SEQ ID NO:54) (1.3 ± 0.8) 0.0001 DC KDR775 (SEQ ID NO:29) 2.2 ± 0.5) 0.0007 DC KDR1084 (SEQ ID NO:46) (1.7 ± 0.3) <0.0001 -
Fig. 17 and Table 8 showed that the formation of tortuous blood vessels was clearly suppressed in the groups vaccinated with DCs pulsed with the peptides of SEQ ID NO: 40 (amino acid initiation position KDR190), SEQ ID NO: 33 (KDR772), SEQ ID NO: 30 (KDR773), SEQ ID NO: 54 (KDR773-2L), SEQ ID NO: 29 (KDR775), and SEQ ID NO: 46 (KDR1084), respectively, as compared to the group to which DC was administered alone.
This indicated a statistically significant angiogenesis inhibitory effect. - Wound healing and fertility in vaccinated mice were analyzed in order to observe the adverse effects of vaccination using these epitope peptides on normal physiological angiogenesis. However, no significant adverse effects were found in the vaccinated mice. Furthermore, using CTL clones that recognize KDR peptides, cytotoxicity against non-proliferative or proliferative endothelial cells was tested in vitro to examine adverse effects in humans. As a result, these clones showed stronger activity against proliferative endothelial cells than against non-proliferative endothelial cells.
- Using A2/Kb transgenic mice expressing human HLA-A0201, the peptides of SEQ ID NO: 30 (amino acid initiation position KDR773) and SEQ ID NO: 34 (KDR1084) were each mixed with incomplete Freund's adjuvant, then administered on
day 3 and 13 to colon cancer strain MC38-implanted tumor-bearing mice so as to indicate changes in tumor volume. - The in vivo anti-tumor effect in a therapeutic model of HLA-A*0201-immobilized epitope peptide (KDR773) was evaluated using the area of the tumor, using the same vaccination method as in Example 22. The result, as apparent from
Fig. 18 , confirm a significant tumor growth suppression effect by vaccination with the peptides of SEQ ID NO: 30 and SEQ ID NO: 34. - The peptide of SEQ ID NO: 40 (amino acid initiation position KDR190) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KWC65-190) obtained from the peptide of SEQ ID NO: 40 in Example 13 was examined using chromium release assay.
- In the results, shown in
Fig. 19 , CTL clone KWC65-190 clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 40. In contrast, absolutely no cytotoxic effect was observed against control cells not presenting the peptide of SEQ ID NO: 40. - The peptide of SEQ ID NO: 33 (amino acid initiation position KDR772) was added to HLA-A0201-positive T2 strain to produce target cells. Using these cells, the cytotoxic effect of the CTL clone (KWC72-772) obtained from the peptide of SEQ ID NO: 33 in Example 13 was examined using a chromium release assay.
- In the results, shown in
Fig. 20 , CTL clone KWC72-772 clearly showed a cytotoxic effect against HLA-A0201-positive cells presenting the peptide of SEQ ID NO: 33. In contrast, absolutely no cytotoxic effect was observed against control cells that did not present the peptide of SEQ ID NO: 33. - The cytotoxic effect of the CTL clone (KWC72-772) against hepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses to express KDR, was examined by chromium release assay. HLA-A0201-positive HePG2 forced to express EGFP was used as control.
- In the results, as shown in
Fig. 21 , CTL clone KWC72-772 showed remarkably high cytotoxic effect against HLA-A0201-positive cells presenting KDR peptides on their surface, as compared to activity against cells presenting the EGFP peptide. - The peptides of SEQ ID NO: 2 (amino acid initiation position KDR1318), SEQ ID NO: 3 (KDR220), SEQ ID NO: 5 (KDR189), and SEQ ID NO: 11 (KDR826) were added to HLA-A24-positive human B-lymphocyte strain A24-LCL (HLA-A24/24, Takara Shuzo) to produce target cells. Using these cells, the cytotoxic effects of CTL clones, C7-1318, KWC46, C18-189, and C65-826, described in Example 3, were examined by chromium release assay.
- In the results, shown in
Figs. 22 to 25 , each of the CTL clones, C7-1318, KWC46, C18-189, and C65-826, clearly showed a cytotoxic effect against each of the HLA-A24-positive cells presenting the peptides of SEQ ID NOs: 2, 3, 5, and 11, respectively. In contrast, hardly any or absolutely no cytotoxic effect was observed against each of the control cells, which did not present the peptides. - As for Example 11, the cytotoxic effects of the CTL clones, C7-1318, KWC46, C18-189, and C65-826, described in Example 3, against human colon cancer HT29 strain (ATCC), forced by adenoviruses to express KDR, were examined by chromium release assay. HLA-A24-positive HT29 strain forcedly expressing EGFP was used as a control.
- In the results, shown in
Figs. 26 to 29 , all CTL clones, C7-1318, KWC46, C18-189, and C65-826, showed remarkably high cytotoxic effect against HLA-A24-positive HT29 forcedly expressing KDR, compared to activity against cells forcedly expressing EGFP. - As for Example 22, the angiogenesis inhibitory activity of peptide epitopes against the angiogenic response induced by
Colon 26 cells in BALB/c mice was evaluated by DAS assay. - As apparent from the results of
Figs. 30 and31 , in the groups vaccinated with DCs pulsed with the peptides of SEQ ID NO: 5 (amino acid initiation position KDR189) and SEQ ID NO: 11 (KDR826), respectively, the formation of tortuous blood vessels was clearly suppressed as compared to the group administered with DC alone. This indicated a statistically significant angiogenesis suppression effect. - The CTL response against each type of following epitope peptides was investigated using PBMC of cancer patients after stimulation with each peptide:
- Peptides of SEQ ID NO: 8 (amino acid initiation position KDR169), SEQ ID NO: 5 (KDR189), SEQ ID NO: 3 (KDR220), SEQ ID NO: 11 (KDR826), and SEQ ID NO: 17 (KDR1318), with high HLA-24 binding affinity; and peptides of SEQ ID NO: 40 (KDR190), SEQ ID NO: 33 (KDR772), SEQ ID NO: 30 (KDR773), SEQ ID NO: 29 (KDR775), and SEQ ID NO: 34 (KDR1328), with high HLA-A0201 binding affinity.
- Using the method reported by Maeda, Y. et al. in Br. J. Cancer 87, 796-804 (2002), peptide-specific CTLs were detected in PBMC. First, PBMC collected from patients (
1x 10 5 cells) were incubated with 10 µM of each peptide in a U-shaped bottom 96-well plate comprising 200 µL medium. The medium consisted of 45% RPMI1640 medium, 45% AIM-V medium, 10% FBS, 100 U/mL interleukin-2 (IL-2), and 0.1 µM MEM non-essential amino acid solution. Every three days, half of the medium was removed and replaced with fresh medium containing the corresponding peptide (20 µM). After incubation for twelve days, cells were collected to test IFN-γ production ability in response to each peptide. When the amount of IFN-γ produced by peptide-stimulated PBMC in response to a corresponding peptide was two times or more than the amount produced in response to the control HIV peptide, that test peptide was deemed positive. - These results showed that a CTL precursor is produced in HLA-A2- and HLA-A24-type cancer patients, as in a healthy donor (Table 9). In the table, patients A, B, and C are HLA-A24-type patients; and patient D is an HLA-A02-type patient. Patients A and D are colon cancer patients; and patients B and C are melanoma patients. CMVs refer to the cytomegalovirus-derived peptides used as positive controls. The peptides with high A-24 binding affinity are described in Kuzushima, K. et al., Blood 2001, 98(6):p1872-1881, and such; and the peptides with high A-2 binding affinity are described in Solache, A. et al., J. of Immunol. 1999, 163(10); p5512-5518, and such.
Table 9 Cancer patient-derived CTL precursor PATIENT
APATIENT
BPATIENT
CPATIENT
DPOSITIVE WELLS TOTAL WELLS POSITIVE WELLS TOTAL WELLS POSITIVE WELLS TOTAL WELLS POSITIVE WELLS TOTAL WELLS CMV 1 32 1 24 0 16 CMV 5 20 KDR 169 12 32 4 32 1 24 KDR 190 3 24 KDR 1893 32 1 32 0 24 KDR 7723 24 KDR 220 0 32 3 32 0 24 KDR 7731 24 KDR 82611 32 3 32 0 24 KDR 7752 24 KDR 1318 0 32 9 32 1 24 KDR 1328 0 24 - As for Example 14 and the like, a standard 51Cr release assay was performed for four hours to establish CTL lines from the CTL precursors (
Fig. 32 ). - The present invention provides novel peptides, which induce cytotoxic T cells by targeting the endothelial cells formed in a wide range of tumoral tissues, and which are extremely effective as cancer vaccines. The present invention also provides pharmaceuticals for treating and preventing tumors, where the pharmaceuticals comprise these peptides.
-
Claims (14)
- A nonapeptide comprising the amino acid sequence of SEQ ID NO: 5.
- A peptide with cytotoxic T cell inducibility, wherein one, two, or more amino acids are substituted or added to the amino acid sequence of SEQ ID NO: 5.
- The peptide of claim 2, wherein the second amino acid from the N terminus is phenylalanine, tyrosine, methionine, or tryptophan.
- The peptide of claim 2 or 3, wherein the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, or methionine.
- A pharmaceutical composition comprising one or more peptides of any one of claims 1 to 4.
- A vaccine comprising a peptide of any one of claims 1 to 4 as an active ingredient.
- A peptide for use in inhibiting angiogenesis at a diseased site, wherein the peptide is selected from group consisting of the peptides of claims 1 to 4.
- A pharmaceutical composition for use in inhibiting angiogenesis at a diseased site, wherein the pharmaceutical comprises one or more peptides of any one of claims 1 to 4.
- An exosome that presents on its surface a complex comprising a peptide of any one of claims 1 to 4, and an HLA antigen.
- The exosome of claim 9, wherein the HLA antigen is HLA-A24.
- The exosome of claim 10, wherein the HLA antigen is HLA-A2402.
- A vaccine for use in inhibiting angiogenesis at a diseased site, wherein the vaccine comprises a peptide of any one of claims 1 to 4 as an active ingredient.
- The peptide of claim 7, the pharmaceutical composition of claim 8, or the vaccine of claim 12, which is used for administration to a subject whose HLA antigen is HLA-A24.
- The peptide, the pharmaceutical composition, or the vaccine of claim 13, which is used for administration to a subject whose HLA antigen is HLA-A2402.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002267285 | 2002-09-12 | ||
JP2003062003 | 2003-03-07 | ||
JP2003167042 | 2003-06-11 | ||
EP03795432A EP1548032B9 (en) | 2002-09-12 | 2003-09-12 | Kdr peptides and vaccines comprising the same |
EP08018875A EP2014678B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
Related Parent Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03795432A Division EP1548032B9 (en) | 2002-09-12 | 2003-09-12 | Kdr peptides and vaccines comprising the same |
EP03795432.8 Division | 2003-09-12 | ||
EP08018875A Division EP2014678B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP08018875.8 Division | 2008-10-29 |
Publications (3)
Publication Number | Publication Date |
---|---|
EP2267023A2 true EP2267023A2 (en) | 2010-12-29 |
EP2267023A3 EP2267023A3 (en) | 2011-02-16 |
EP2267023B1 EP2267023B1 (en) | 2015-02-11 |
Family
ID=31998762
Family Applications (11)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10010169.0A Expired - Lifetime EP2267021B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP08018875A Expired - Lifetime EP2014678B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP08018897A Expired - Lifetime EP2014679B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010174.0A Expired - Lifetime EP2267022B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010173.2A Expired - Lifetime EP2261249B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010171A Withdrawn EP2261247A3 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010176.5A Expired - Lifetime EP2270042B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010170.8A Expired - Lifetime EP2270041B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010172.4A Expired - Lifetime EP2261248B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP03795432A Expired - Lifetime EP1548032B9 (en) | 2002-09-12 | 2003-09-12 | Kdr peptides and vaccines comprising the same |
EP10010175.7A Expired - Lifetime EP2267023B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
Family Applications Before (10)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10010169.0A Expired - Lifetime EP2267021B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP08018875A Expired - Lifetime EP2014678B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP08018897A Expired - Lifetime EP2014679B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010174.0A Expired - Lifetime EP2267022B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010173.2A Expired - Lifetime EP2261249B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010171A Withdrawn EP2261247A3 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010176.5A Expired - Lifetime EP2270042B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010170.8A Expired - Lifetime EP2270041B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP10010172.4A Expired - Lifetime EP2261248B1 (en) | 2002-09-12 | 2003-09-12 | KDR peptides and vaccines comprising the same |
EP03795432A Expired - Lifetime EP1548032B9 (en) | 2002-09-12 | 2003-09-12 | Kdr peptides and vaccines comprising the same |
Country Status (14)
Country | Link |
---|---|
US (5) | US7514084B2 (en) |
EP (11) | EP2267021B1 (en) |
JP (3) | JP3971769B2 (en) |
CN (5) | CN103073620B (en) |
AT (2) | ATE432291T1 (en) |
AU (1) | AU2003264419A1 (en) |
CY (2) | CY1109463T1 (en) |
DE (1) | DE60327786D1 (en) |
DK (6) | DK2267022T3 (en) |
ES (2) | ES2375299T3 (en) |
HK (7) | HK1124077A1 (en) |
PT (2) | PT1548032E (en) |
SI (2) | SI2014678T1 (en) |
WO (1) | WO2004024766A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8975229B2 (en) | 2009-06-11 | 2015-03-10 | Oncotherapy Science, Inc. | Methods for treating a disease caused by choroidal neovascularization |
Families Citing this family (68)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103073620B (en) * | 2002-09-12 | 2014-12-10 | 肿瘤疗法科学股份有限公司 | KDR peptides and vaccines comprising the same |
US9090673B2 (en) * | 2003-12-12 | 2015-07-28 | City Of Hope | Synthetic conjugate of CpG DNA and T-help/CTL peptide |
JP4628208B2 (en) * | 2004-08-10 | 2011-02-09 | オンコセラピー・サイエンス株式会社 | Peptide vaccine for the treatment of gastric cancer or colorectal cancer expressing CXADRL1 or GCUD1 protein |
DK2325306T3 (en) * | 2005-02-25 | 2014-03-03 | Oncotherapy Science Inc | Peptide vaccines against lung cancers expressing TTK, URLC10 or KOC1 polypeptide |
EP2289533B1 (en) | 2005-02-28 | 2013-09-18 | Oncotherapy Science, Inc. | Epitope peptides derived from vascular endothelial growth factor receptor 1 and vaccines containing these peptides |
US20060234941A1 (en) | 2005-04-15 | 2006-10-19 | The Gov. Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services | Peptide epitopes of VEGFR-2/KDR that inhibit angiogenesis |
WO2007013480A2 (en) | 2005-07-29 | 2007-02-01 | Oncotherapy Science, Inc. | Screening and therapeutic method for nsclc targeting cdca1-kntc2 complex |
ATE534415T1 (en) | 2005-12-13 | 2011-12-15 | Harvard College | SCAFFOLDS FOR CELL TRANSPLANTATION |
PL2059256T3 (en) | 2006-08-11 | 2017-02-28 | Life Sciences Research Partners Vzw | Immunogenic peptides and their use in immune disorders |
TWI441648B (en) * | 2007-01-03 | 2014-06-21 | Oncotherapy Science Inc | Foxp3 peptide vaccine |
KR101493775B1 (en) * | 2007-02-16 | 2015-02-16 | 온코세라피 사이언스 가부시키가이샤 | Vaccine therapy for choroidal neovascularization |
TWI434853B (en) * | 2007-04-11 | 2014-04-21 | Oncotherapy Science Inc | Tem8 peptides and vaccines comprising the same |
TW200908998A (en) * | 2007-06-27 | 2009-03-01 | Oncotherapy Science Inc | Compositions and methods of treating cancer |
MX2010002002A (en) | 2007-08-20 | 2010-03-11 | Oncotherapy Science Inc | Foxm1 peptide and medicinal agent comprising the same. |
BRPI0815578B8 (en) * | 2007-08-20 | 2021-05-25 | Oncotherapy Science Inc | peptides, pharmaceutical agents comprising the same, in vitro methods for inducing an antigen-presenting cell, uses of said peptides, and vaccine |
TWI436775B (en) * | 2007-08-24 | 2014-05-11 | Oncotherapy Science Inc | Combination therapy for pancreatic cancer using an antigenic peptide and chemotherapeutic agent |
TW200932260A (en) * | 2007-11-28 | 2009-08-01 | Oncotherapy Science Inc | STAT3 epitope peptides |
JP5690143B2 (en) | 2008-02-13 | 2015-03-25 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Continuous cell programming device |
EP2245059B1 (en) | 2008-02-14 | 2017-09-20 | Life Sciences Research Partners VZW | Cd4+ t-cells with cytolytic properties |
ES2676630T3 (en) | 2008-02-14 | 2018-07-23 | Life Sciences Research Partners Vzw | Immunogenic control of tumors and tumor cells |
ES2545885T3 (en) | 2008-02-14 | 2015-09-16 | Life Sciences Research Partners Vzw | Elimination of immune responses against viral vectors |
EP2719396B1 (en) | 2008-02-14 | 2017-06-21 | Life Sciences Research Partners VZW | Immunotherapy targeting intracellular pathogens |
TW201000119A (en) * | 2008-06-10 | 2010-01-01 | Oncotherapy Science Inc | MYBL2 epitope peptides and vaccines containing the same |
TWI526219B (en) * | 2008-06-19 | 2016-03-21 | 腫瘤療法 科學股份有限公司 | Cdca1 epitope peptides and vaccines containing the same |
AU2009292643B2 (en) | 2008-09-19 | 2016-02-18 | Nektar Therapeutics | Polymer conjugates of therapeutic peptides |
US8883966B2 (en) | 2008-10-22 | 2014-11-11 | Oncotherapy Science, Inc. | RAB6KIFL/KIF20A epitope peptide and vaccines containing the same |
TWI539160B (en) | 2008-12-05 | 2016-06-21 | 腫瘤療法 科學股份有限公司 | Wdrpuh epitope peptides and vaccines containing the same |
TW201032820A (en) * | 2008-12-16 | 2010-09-16 | Oncotherapy Science Inc | ELOVL7 epitope peptides and vaccines containing the same |
CN102333867B (en) * | 2008-12-24 | 2015-01-14 | 肿瘤疗法科学股份有限公司 | C1orf59 peptides and vaccines including the same |
EP2684957A2 (en) | 2009-01-08 | 2014-01-15 | International Institute of Cancer Immunology, Inc. | Double-stranded siRNA targeting cancer antigen eEF2 |
BRPI1012312A2 (en) * | 2009-03-04 | 2017-03-21 | Oncotherapy Science Inc | vangl1 peptides and vaccines including the same |
WO2010106770A1 (en) * | 2009-03-18 | 2010-09-23 | Oncotherapy Science, Inc. | Neil3 peptides and vaccines including the same |
TW201100090A (en) * | 2009-04-01 | 2011-01-01 | Oncotherapy Science Inc | C6orf167 peptides and vaccines containing the same |
TWI507204B (en) * | 2009-05-26 | 2015-11-11 | Oncotherapy Science Inc | Cdc45l peptides and vaccines including the same |
EP2461828B1 (en) | 2009-07-31 | 2017-06-21 | President and Fellows of Harvard College | Programming of cells for tolerogenic therapies |
TW201200525A (en) * | 2009-12-04 | 2012-01-01 | Oncotherapy Science Inc | MYBL2 peptides and vaccines containing the same |
TW201136604A (en) * | 2009-12-14 | 2011-11-01 | Oncotherapy Science Inc | TMEM22 peptides and vaccines including the same |
CN102115492B (en) * | 2010-01-05 | 2013-03-06 | 中国农业科学院北京畜牧兽医研究所 | Vascular endothelial growth factor receptor partial polypeptide and application thereof |
AU2011225577B2 (en) | 2010-03-11 | 2017-01-19 | Oncotherapy Science, Inc. | HJURP peptides and vaccines including the same |
TW201134480A (en) * | 2010-04-08 | 2011-10-16 | Oncotherapy Science Inc | CLUAP1 peptides and vaccines including the same |
RU2012147590A (en) * | 2010-04-09 | 2014-05-20 | Онкотерапи Сайенс, Инк. | CDCA5 PEPTIDES AND THEIR VACCINES CONTAINING THEM |
TW201211250A (en) * | 2010-09-07 | 2012-03-16 | Oncotherapy Science Inc | VANGL1 peptides and vaccines including the same |
KR102155383B1 (en) | 2010-10-06 | 2020-09-11 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Injectable, pore-forming hydrogels for materials-based cell therapies |
CA2820617C (en) | 2010-11-25 | 2020-01-07 | Imnate Sarl | Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination |
KR102004900B1 (en) | 2010-12-02 | 2019-07-29 | 온코세라피 사이언스 가부시키가이샤 | Tomm34 peptides and vaccines including the same |
GB201201511D0 (en) | 2012-01-30 | 2012-03-14 | Univ Leuven Kath | Modified epitopes for boosting CD4+ T-cell responses |
SG11201405578TA (en) | 2012-03-09 | 2014-11-27 | Oncotherapy Science Inc | Pharmaceutical composition containing peptide |
US9937249B2 (en) | 2012-04-16 | 2018-04-10 | President And Fellows Of Harvard College | Mesoporous silica compositions for modulating immune responses |
JP6263117B2 (en) | 2012-05-11 | 2018-01-17 | 公益財団法人微生物化学研究会 | Anti-CXADR antibody |
EP2872532A4 (en) | 2012-07-10 | 2016-04-13 | Oncotherapy Science Inc | Cdca1 epitope peptides for th1 cells and vaccines containing the same |
AU2013356143A1 (en) * | 2012-12-04 | 2015-06-04 | Oncotherapy Science, Inc. | SEMA5B peptides and vaccines containing the same |
EP2955226B1 (en) | 2013-02-08 | 2019-06-26 | Medical & Biological Laboratories Co., Ltd. | Antibodies to human nrg1 protein |
GB201309469D0 (en) | 2013-05-28 | 2013-07-10 | Imcyse Sa | Detection of CD4+ T lymphocytes |
JP2017042047A (en) * | 2013-12-06 | 2017-03-02 | オンコセラピー・サイエンス株式会社 | Vegfr2-derived peptide and vaccine comprising the same |
CN107073090A (en) | 2014-04-30 | 2017-08-18 | 哈佛学院董事会 | With reference to vaccine device and kill cancer cell method |
GB201418433D0 (en) | 2014-10-17 | 2014-12-03 | Imcyse Sa | Novel immunogenic peptides |
WO2016123573A1 (en) | 2015-01-30 | 2016-08-04 | President And Fellows Of Harvard College | Peritumoral and intratumoral materials for cancer therapy |
WO2016129335A1 (en) * | 2015-02-09 | 2016-08-18 | 株式会社ライフアートビレッジ | Method for screening for peptide for use in immune cell therapy |
US11150242B2 (en) | 2015-04-10 | 2021-10-19 | President And Fellows Of Harvard College | Immune cell trapping devices and methods for making and using the same |
US10729791B2 (en) | 2015-05-18 | 2020-08-04 | Imcyse Sa | Animal models for evaluating pharmaceutical compounds |
EP3939606A3 (en) | 2015-09-25 | 2022-04-20 | ImCyse SA | Improved methods and compounds for eliminating immune responses to therapeutic agents |
CN105200010B (en) * | 2015-10-15 | 2019-02-12 | 首都医科大学附属北京佑安医院 | The extracorporeal culturing method and its special culture media of People living with HIV HLA-A2 Specific CTL Cells |
CN115531609A (en) | 2016-02-06 | 2022-12-30 | 哈佛学院校长同事会 | Remodeling hematopoietic niches to reconstitute immunity |
SG11201808710UA (en) | 2016-04-19 | 2018-11-29 | Imcyse Sa | Novel immunogenic cd1d binding peptides |
MA45738A (en) | 2016-07-13 | 2019-05-22 | Harvard College | MIMETIC SCAFFOLDING OF ANTIGEN PRESENT CELLS AND METHODS FOR PREPARING AND USING THEM |
JP7168228B2 (en) * | 2017-05-19 | 2022-11-09 | 慶應義塾 | Peptide vaccine and peptide vaccine composition for cranial nerve disease |
CN111363026B (en) * | 2020-03-31 | 2022-03-25 | 浙江省中医院、浙江中医药大学附属第一医院(浙江省东方医院) | Method for enhancing affinity and stability of antigen polypeptide |
KR20220022021A (en) * | 2020-08-14 | 2022-02-23 | 서울대학교산학협력단 | A pharmaceutical composition for preventing or treating cancer comprising hepatitis b virus-derived polypeptide |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5861301A (en) | 1992-02-20 | 1999-01-19 | American Cayanamid Company | Recombinant kinase insert domain containing receptor and gene encoding same |
JPH11510507A (en) | 1995-08-03 | 1999-09-14 | リュークスウニベルジテート トゥ ライデン | Cell-derived antigen-presenting vesicles |
WO1999067288A1 (en) | 1998-06-25 | 1999-12-29 | Sumitomo Pharmaceuticals Company, Limited | Tumor antigen peptides originating in cyclophilin b |
JP2000509281A (en) | 1996-04-30 | 2000-07-25 | デューク ユニバーシティ | Method for treating cancer and pathogen infection using antigen-presenting cells to which RNA has been added |
JP2000512161A (en) | 1997-07-16 | 2000-09-19 | アンスティチュ・ナショナル・ドゥ・ラ・サンテ・エ・ドゥ・ラ・ルシェルシュ・メディカル | Intracellular vesicles called "exosomes", their use in preparation and immunostimulation |
JP2002267285A (en) | 2001-03-12 | 2002-09-18 | Denso Corp | Cooler/refrigerator |
JP2003062003A (en) | 2001-06-13 | 2003-03-04 | Topcon Corp | Microscope for surgery |
JP2003167042A (en) | 2001-11-30 | 2003-06-13 | Nec Corp | Cellular phone terminal having position measuring function |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6222012B1 (en) * | 1992-08-31 | 2001-04-24 | Ludwig Institute For Cancer Research | Isolated nonapeptides presented by HLA molecules, and uses thereof |
CN1701814A (en) * | 1992-11-13 | 2005-11-30 | 马克斯普朗克科学促进协会 | F1K-1 is a receptor for vascular endothelial growth factor |
CA2158745C (en) * | 1993-03-25 | 2007-06-19 | Richard L. Kendall | Inhibitor of vascular endothelial cell growth factor |
US6100071A (en) * | 1996-05-07 | 2000-08-08 | Genentech, Inc. | Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production |
US6080728A (en) * | 1996-07-16 | 2000-06-27 | Mixson; A. James | Carrier: DNA complexes containing DNA encoding anti-angiogenic peptides and their use in gene therapy |
US5942428A (en) * | 1996-08-21 | 1999-08-24 | Sugen, Inc. | Crystals of the tyrosine kinase domain of non-insulin receptor tyrosine kinases |
DE19638745C2 (en) | 1996-09-11 | 2001-05-10 | Schering Ag | Monoclonal antibodies against the extracellular domain of the human VEGF receptor protein (KDR) |
JP3837748B2 (en) * | 1997-01-17 | 2006-10-25 | 東亞合成株式会社 | VEGF binding polypeptide |
ES2292204T3 (en) * | 1997-06-18 | 2008-03-01 | MERCK & CO., INC. (A NEW JERSEY CORP.) | HUMAN TYPE CINASE THIROSINE RECEPTOR, KDR. |
WO1999029715A1 (en) * | 1997-12-05 | 1999-06-17 | Kyogo Itoh | Tumor antigen peptide derivatives |
AU2299099A (en) | 1998-02-04 | 1999-08-23 | Kyowa Hakko Kogyo Co. Ltd. | Antibodies against human vegf receptor kdr |
GB9804121D0 (en) * | 1998-02-26 | 1998-04-22 | Cancer Res Campaign Tech | Anti-angiogenic vaccines: materials and methods relating thereto |
EP1086705A4 (en) * | 1998-05-20 | 2002-02-06 | Kyowa Hakko Kogyo Kk | Vegf activity inhibitors |
WO2001012809A2 (en) * | 1999-08-13 | 2001-02-22 | Crucell Holland B.V. | Heterodimeric vegf variants used for inhibiting angiogenesis |
US7264810B2 (en) | 2001-01-19 | 2007-09-04 | Cytos Biotechnology Ag | Molecular antigen array |
CU23178A1 (en) * | 2002-04-15 | 2006-09-22 | Ct Ingenieria Genetica Biotech | ANTIANGIOGEN ACTIVE ACTIVE IMMUNOTHERAPY |
CN103073620B (en) * | 2002-09-12 | 2014-12-10 | 肿瘤疗法科学股份有限公司 | KDR peptides and vaccines comprising the same |
WO2004027027A2 (en) | 2002-09-18 | 2004-04-01 | Trustees Of The University Of Pennsylvania | Method of inhibiting choroidal neovascularization |
EP2289533B1 (en) | 2005-02-28 | 2013-09-18 | Oncotherapy Science, Inc. | Epitope peptides derived from vascular endothelial growth factor receptor 1 and vaccines containing these peptides |
KR101493775B1 (en) | 2007-02-16 | 2015-02-16 | 온코세라피 사이언스 가부시키가이샤 | Vaccine therapy for choroidal neovascularization |
TWI436775B (en) | 2007-08-24 | 2014-05-11 | Oncotherapy Science Inc | Combination therapy for pancreatic cancer using an antigenic peptide and chemotherapeutic agent |
-
2003
- 2003-09-12 CN CN201210430723.6A patent/CN103073620B/en not_active Expired - Fee Related
- 2003-09-12 EP EP10010169.0A patent/EP2267021B1/en not_active Expired - Lifetime
- 2003-09-12 PT PT03795432T patent/PT1548032E/en unknown
- 2003-09-12 PT PT08018875T patent/PT2014678E/en unknown
- 2003-09-12 EP EP08018875A patent/EP2014678B1/en not_active Expired - Lifetime
- 2003-09-12 EP EP08018897A patent/EP2014679B1/en not_active Expired - Lifetime
- 2003-09-12 EP EP10010174.0A patent/EP2267022B1/en not_active Expired - Lifetime
- 2003-09-12 WO PCT/JP2003/011722 patent/WO2004024766A1/en active Application Filing
- 2003-09-12 ES ES08018875T patent/ES2375299T3/en not_active Expired - Lifetime
- 2003-09-12 DK DK10010174.0T patent/DK2267022T3/en active
- 2003-09-12 DE DE60327786T patent/DE60327786D1/en not_active Expired - Lifetime
- 2003-09-12 CN CNB038250810A patent/CN100352843C/en not_active Expired - Lifetime
- 2003-09-12 ES ES03795432T patent/ES2327040T3/en not_active Expired - Lifetime
- 2003-09-12 CN CN2007101802067A patent/CN101139393B/en not_active Expired - Fee Related
- 2003-09-12 EP EP10010173.2A patent/EP2261249B1/en not_active Expired - Lifetime
- 2003-09-12 EP EP10010171A patent/EP2261247A3/en not_active Withdrawn
- 2003-09-12 SI SI200332101T patent/SI2014678T1/en unknown
- 2003-09-12 EP EP10010176.5A patent/EP2270042B1/en not_active Expired - Lifetime
- 2003-09-12 DK DK10010175.7T patent/DK2267023T3/en active
- 2003-09-12 EP EP10010170.8A patent/EP2270041B1/en not_active Expired - Lifetime
- 2003-09-12 US US10/527,496 patent/US7514084B2/en not_active Expired - Fee Related
- 2003-09-12 EP EP10010172.4A patent/EP2261248B1/en not_active Expired - Lifetime
- 2003-09-12 CN CN201410214247.3A patent/CN103951745A/en active Pending
- 2003-09-12 DK DK10010169.0T patent/DK2267021T3/en active
- 2003-09-12 AT AT03795432T patent/ATE432291T1/en active
- 2003-09-12 AT AT08018875T patent/ATE531729T1/en active
- 2003-09-12 DK DK08018875.8T patent/DK2014678T3/en active
- 2003-09-12 DK DK10010173.2T patent/DK2261249T3/en active
- 2003-09-12 JP JP2004571938A patent/JP3971769B2/en not_active Expired - Lifetime
- 2003-09-12 AU AU2003264419A patent/AU2003264419A1/en not_active Abandoned
- 2003-09-12 EP EP03795432A patent/EP1548032B9/en not_active Expired - Lifetime
- 2003-09-12 CN CN2007101802052A patent/CN101139392B/en not_active Expired - Fee Related
- 2003-09-12 DK DK03795432T patent/DK1548032T3/en active
- 2003-09-12 SI SI200331649T patent/SI1548032T1/en unknown
- 2003-09-12 EP EP10010175.7A patent/EP2267023B1/en not_active Expired - Lifetime
-
2007
- 2007-03-08 JP JP2007058127A patent/JP4185143B2/en not_active Expired - Fee Related
- 2007-05-08 JP JP2007123850A patent/JP4185147B2/en not_active Expired - Fee Related
-
2009
- 2009-01-16 US US12/355,616 patent/US7695720B2/en not_active Expired - Lifetime
- 2009-04-30 HK HK09104058.2A patent/HK1124077A1/en not_active IP Right Cessation
- 2009-05-15 HK HK11104992.7A patent/HK1151036A1/en not_active IP Right Cessation
- 2009-05-15 HK HK11104996.3A patent/HK1151039A1/en not_active IP Right Cessation
- 2009-05-15 HK HK09104475.7A patent/HK1125659A1/en not_active IP Right Cessation
- 2009-05-15 HK HK11104995.4A patent/HK1151038A1/en not_active IP Right Cessation
- 2009-05-15 HK HK11104977.6A patent/HK1151032A1/en not_active IP Right Cessation
- 2009-05-15 HK HK11104993.6A patent/HK1151037A1/en not_active IP Right Cessation
- 2009-08-12 CY CY20091100856T patent/CY1109463T1/en unknown
-
2010
- 2010-02-23 US US12/711,159 patent/US8206719B2/en not_active Expired - Fee Related
-
2012
- 2012-01-19 CY CY20121100072T patent/CY1113379T1/en unknown
- 2012-06-12 US US13/494,930 patent/US8574585B2/en not_active Expired - Fee Related
- 2012-06-12 US US13/494,933 patent/US8574586B2/en not_active Expired - Fee Related
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5861301A (en) | 1992-02-20 | 1999-01-19 | American Cayanamid Company | Recombinant kinase insert domain containing receptor and gene encoding same |
JPH11510507A (en) | 1995-08-03 | 1999-09-14 | リュークスウニベルジテート トゥ ライデン | Cell-derived antigen-presenting vesicles |
JP2000509281A (en) | 1996-04-30 | 2000-07-25 | デューク ユニバーシティ | Method for treating cancer and pathogen infection using antigen-presenting cells to which RNA has been added |
JP2000512161A (en) | 1997-07-16 | 2000-09-19 | アンスティチュ・ナショナル・ドゥ・ラ・サンテ・エ・ドゥ・ラ・ルシェルシュ・メディカル | Intracellular vesicles called "exosomes", their use in preparation and immunostimulation |
WO1999067288A1 (en) | 1998-06-25 | 1999-12-29 | Sumitomo Pharmaceuticals Company, Limited | Tumor antigen peptides originating in cyclophilin b |
JP2002267285A (en) | 2001-03-12 | 2002-09-18 | Denso Corp | Cooler/refrigerator |
JP2003062003A (en) | 2001-06-13 | 2003-03-04 | Topcon Corp | Microscope for surgery |
JP2003167042A (en) | 2001-11-30 | 2003-06-13 | Nec Corp | Cellular phone terminal having position measuring function |
Non-Patent Citations (29)
Title |
---|
"Development of Pharmaceuticals, New Series", PEPTIDE SYNTHESIS, vol. 14, 1991 |
"Foundations and Experiments in Peptide Synthesis", 1985, MARUZEN CO., LTD. |
"Peptide Synthesis", 1975, MARUZEN CO., LTD. |
"Peptide Synthesis, Interscience", 1966 |
"The Proteins", vol. 2, 1976, ACADEMIC PRESS INC. |
BENMOHAMED L.; KRISHNAN R.; LONGMATE J.; AUGE C.; LOW L.; PRIMUS J.; DIAMOND DJ.: "Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DRl transgenic mice: dependence on HLA class II restricted T(H) response.", HUM. IMMUNOL., vol. 61, no. 8, August 2000 (2000-08-01), pages 764 - 79 |
BICKNELL, R.; HARRIS, A.L., CURR. OPIN. ONCOL., vol. 8, 1996, pages 60 - 65 |
CANCER RES., vol. 56, 1996, pages 5672 |
CANCER RESEARCH, vol. 62, 1 November 2002 (2002-11-01), pages 6116 - 6123 |
CLIN. MICROBIOL. REV., vol. 7, 1994, pages 277 - 289 |
CLINICAL CANCER RESEARCH, vol. 5, August 1999 (1999-08-01), pages 2185 - 2191 |
FOLKMAN, J., NATURE MED., vol. 1, 1995, pages 27 - 31 |
HUM. IMMUNOL., vol. 61, no. 8, August 2000 (2000-08-01), pages 764 - 79 |
IMMUNOGENETICS., vol. 41, 1995, pages 178 |
J. BIOL. CHEM., vol. 269, no. 43, 28 October 1994 (1994-10-28), pages 26988 - 95 |
J. EXP. MED., vol. 184, 1996, pages 465 |
J. IMMUNOL., vol. 152, 1994, pages 3913 |
J. IMMUNOL., vol. 155, 1994, pages 4307 |
J. IRNMUNOL., vol. 161, 1998, pages 5607 |
KUZUSLUMA, K. ET AL., BLOOD, vol. 98, no. 6, 2001, pages I872 - 1881 |
LEUKEMIA, vol. 13, no. 4, April 1999 (1999-04-01), pages 605 - 13 |
MAEDA, Y. ET AL., BR. J. CANCER, vol. 87, 2002, pages 796 - 804 |
MIYAKE, S. ET AL., PROC. NATL. ACAD. SCI. USA., vol. 93, 1996, pages 1320 - 1324 |
NUKAYA, I., INT. J. CANCER, vol. 80, 1999 |
PARKER K. C., J. IMMUNOL., vol. 152, 1994 |
PARKER, K. C., J. IMMUNOL., vol. 152, 1994 |
SOLACHE, A. ET AL., J. OF IMMUNOL., vol. 163, no. 10, 1999, pages 5512 - 5518 |
TANAKA, H. ET AL., AACR #3669, vol. 42, March 2001 (2001-03-01), pages 681 - 682 |
THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 195, no. 12, 2002, pages 1575 - 1584 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8975229B2 (en) | 2009-06-11 | 2015-03-10 | Oncotherapy Science, Inc. | Methods for treating a disease caused by choroidal neovascularization |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2014679B1 (en) | KDR peptides and vaccines comprising the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AC | Divisional application: reference to earlier application |
Ref document number: 1548032 Country of ref document: EP Kind code of ref document: P Ref document number: 2014678 Country of ref document: EP Kind code of ref document: P |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR |
|
PUAL | Search report despatched |
Free format text: ORIGINAL CODE: 0009013 |
|
AK | Designated contracting states |
Kind code of ref document: A3 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR |
|
17P | Request for examination filed |
Effective date: 20110811 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1151038 Country of ref document: HK |
|
17Q | First examination report despatched |
Effective date: 20120709 |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
INTG | Intention to grant announced |
Effective date: 20140806 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AC | Divisional application: reference to earlier application |
Ref document number: 1548032 Country of ref document: EP Kind code of ref document: P Ref document number: 2014678 Country of ref document: EP Kind code of ref document: P |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: DK Ref legal event code: T3 Effective date: 20150217 |
|
REG | Reference to a national code |
Ref country code: SE Ref legal event code: TRGR |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 709879 Country of ref document: AT Kind code of ref document: T Effective date: 20150315 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 60347305 Country of ref document: DE Effective date: 20150326 |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: VDEP Effective date: 20150211 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 709879 Country of ref document: AT Kind code of ref document: T Effective date: 20150211 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1151038 Country of ref document: HK |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150512 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 Ref country code: NL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 60347305 Country of ref document: DE |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
26N | No opposition filed |
Effective date: 20151112 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R119 Ref document number: 60347305 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: DK Ref legal event code: EBP Effective date: 20150930 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150912 Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
REG | Reference to a national code |
Ref country code: SE Ref legal event code: EUG |
|
GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20150912 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150913 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: MM4A |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: ST Effective date: 20160531 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150930 Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150912 Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150930 Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160401 Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150912 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150930 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DK Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150930 Ref country code: BG Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160630 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20030912 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150930 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150211 |